T cell receptors and immune therapy using the same against prame positive cancers

ABSTRACT

The present invention pertains to antigen recognizing constructs against tumor associated antigens (TAA), in particular against Preferentially Expressed Antigen of Melanoma (PRAME). The invention in particular provides novel T cell receptor (TCR) based molecules which are selective and specific for the tumor expressed antigen of the invention. The TCR of the invention, and TAA binding fragments derived therefrom, are of use for the diagnosis, treatment and prevention of TAA expressing cancerous diseases. Further provided are nucleic acids encoding the antigen recognizing constructs of the invention, vectors comprising these nucleic acids, recombinant cells expressing the antigen recognizing constructs and pharmaceutical compositions comprising the compounds of the invention.

CROSS REFERENCE TO RELATED APPLICATIONS

This is a continuation application of U.S. patent application Ser. No.15/928,785, filed 22 Mar. 2018, which claims the benefit of U.S.Provisional Application Ser. No. 62/475,329, filed 23 Mar. 2017, andGerman Application No. 102017106305.6, filed 23 Mar. 2017, the contentof each of these applications is herein incorporated by reference intheir entirety.

Pursuant to the EFS-Web legal framework and 37 CFR §§ 1.821-825 (seeMPEP § 2442.03(a)), a Sequence Listing in the form of an ASCII-complianttext file (entitled “Sequence_Listing_3000058-008002_ST25.txt” createdon 9 Apr. 2019, and 116,097 bytes in size) is submitted concurrentlywith the instant application, and the entire contents of the SequenceListing are incorporated herein by reference.

The present invention pertains to antigen recognizing constructs againsttumor associated antigens (TAA), in particular against PreferentiallyExpressed Antigen of Melanoma (PRAME). The invention in particularprovides novel T cell receptor (TCR) based molecules which are selectiveand specific for the tumor expressed antigen of the invention. The TCRof the invention, and TAA binding fragments derived therefrom, are ofuse for the diagnosis, treatment and prevention of TAA expressingcancerous diseases. Further provided are nucleic acids encoding theantigen recognizing constructs of the invention, vectors comprisingthese nucleic acids, recombinant cells expressing the antigenrecognizing constructs and pharmaceutical compositions comprising thecompounds of the invention.

DESCRIPTION

PRAME is encoded by the PRAME gene, which is expressed at a high levelin a large proportion of tumors, including melanomas, non-small-celllung carcinomas, ovarian carcinoma renal cell carcinoma (RCC), breastcarcinoma, cervix carcinoma, colon carcinoma, sarcoma, neuroblastoma, aswell as several types of leukemia. PRAME is the best characterizedmember of the PRAME family of leucine-rich repeat (LRR) proteins.Mammalian genomes contain multiple members of the PRAME family whereasin other vertebrate genomes only one PRAME-like LRR protein wasidentified. PRAME is a cancer/testis antigen that is expressed at verylow levels in normal adult tissues except testis but at high levels in avariety of cancer cells.

T-cell based immunotherapy targets represent peptide epitopes derivedfrom tumor-associated or tumor-specific proteins, which are presented bymolecules of the major histocompatibility complex (MHC). These tumorsassociated antigens (TAAs) can be peptides derived from all proteinclasses, such as enzymes, receptors, transcription factors, etc. whichare expressed and, as compared to unaltered cells of the same origin,usually up-regulated in cells of the respective tumor.

Specific elements of the cellular immune response are capable ofspecifically recognizing and destroying tumor cells. The isolation ofT-cells from tumor-infiltrating cell populations or from peripheralblood suggests that such cells play an important role in natural immunedefense against cancer. CD8-positive T-cells in particular, whichrecognize class I molecules of the major histocompatibility complex(MHC)-bearing peptides of usually 8 to 10 amino acid residues derivedfrom proteins or defective ribosomal products (DRiPs) located in thecytosol, play an important role in this response. The MHC-molecules ofthe human are also designated as human leukocyte-antigens (HLA).

There are two classes of MHC-molecules, MHC class I and MHC class II.Complexes of peptide and MHC class I are recognized by CD8-positiveT-cells bearing the appropriate T-cell receptor (TCR), whereas complexesof peptide and MHC class II molecules are recognized byCD4-positive-helper-T-cells bearing the appropriate TCR. Since bothtypes of response, CD8 and CD4 dependent, contribute jointly andsynergistically to the anti-tumor effect, the identification andcharacterization of tumor-associated antigens and corresponding T cellreceptors is important in the development of cancer immunotherapies suchas vaccines and cell therapies.

In the MHC class I dependent immune reaction, peptides not only have tobe able to bind to certain MHC class I molecules expressed by tumorcells, they subsequently also have to be recognized by T-cells bearingspecific T-cell receptors (TCR). Therefore, TAAs are a starting pointfor the development of a T-cell based therapy including but not limitedto tumor vaccines and cell therapies.

Approximately 90 percent of peripheral blood T cells express a TCRconsisting of an α poly-peptide and a β polypeptide. A small percentageof T cells (about 5% of total T cells) have been shown to express a TCRconsisting of a γ polypeptide and δ polypeptide. γδ T cells are found attheir highest abundance in the gut mucosa, within a population oflymphocytes known as intraepithelial lymphocytes (IELs). The antigenicmolecules that activate γδ T cells are still widely unknown. However, γδT cells are not MHC restricted and seem to be able to recognize wholeproteins rather than requiring peptides to be presented by MHC moleculeson antigen presenting cells, although some recognize MHC class IBmolecules. Human Vγ9/Vδ2 T cells, which constitute the major γδ T cellpopulation in peripheral blood, are unique in that they specifically andrapidly respond to a small non-peptidic microbial metabolite, HMB-PP, anisopentenyl pyrophosphate precursor. Estimates of the percentages of Tcells that may be found in peripheral blood from healthy donors are asfollows: CD3+=70.78%±4.71; CD3+CD4+=38.97%±5.66; CD3+CD8+=28.955%±7.43;CD3+CD56+=5.22%±1.74, CD3−CD56+=10.305%±4.7, CD3+CD45RA+=45.00%±7.19,and CD3+CD45RO+=27.21%±7.34.

The chains of the T cell antigen receptor of a T cell clone are eachcomposed of a unique combination of domains designated variable (V),[diversity (D),] joining (J), and constant (C). In each T cell clone,the combination of V, D and J domains of both the alpha and the betachains or of both the delta and gamma chains participates in antigenrecognition in a manner which is uniquely characteristic of that T cellclone and defines a unique binding site, also known as the idiotype ofthe T cell clone. In contrast, the C domain does not participate inantigen binding.

A TCR is a heterodimeric cell surface protein of the immunoglobulinsuper-family, which is associated with invariant proteins of the CD3complex involved in mediating signal transduction. TCRs exist in αβ andγδ forms, which are structurally similar but have quite distinctanatomical locations and probably functions. The extracellular portionof native heterodimeric αβTCR and γδTCR each contain two polypeptides,each of which has a membrane-proximal constant domain, and amembrane-distal variable domain. Each of the constant and variabledomains include an intra-chain disulfide bond. The variable domainscontain the highly polymorphic loops analogous to the complementaritydetermining regions (CDRs) of antibodies. The use of TCR gene therapyovercomes a number of current hurdles. It allows equipping patients' ownT cells with desired specificities and generation of sufficient numbersof T cells in a short period of time, avoiding their exhaustion. The TCRwill be transduced into potent T cells (e.g. central memory T cells or Tcells with stem cell characteristics), which may ensure betterpersistence and function upon transfer. TCR-engineered T cells will beinfused into cancer patients rendered lymphopenic by chemotherapy orirradiation, allowing efficient engraftment but inhibiting immunesuppression.

While advances have been made in the development of molecular-targetingdrugs for cancer therapy, there remains a need in the art to develop newanti-cancer agents that specifically target molecules highly specific tocancer cells. The present description addresses that need by providingnovel PRAME TCRs, respective recombinant TCR constructs, nucleic acids,vectors and host cells that specifically bind TAA epitope(s) asdisclosed; and methods of using such molecules in the treatment ofcancer. The term TAA in context of the invention relates in particularto the following preferred proteins: PRAME, and fragments or analogsthereof, in particular fragments or analogs comprising or consisting ofthe antigenic peptide sequences shown in SEQ ID NO: 97 to 115,preferably SEQ ID NO: 97 to 106, more preferably SEQ ID NO: 97.

The object of the invention is solved in a first aspect by an antigenrecognizing construct comprising at least one complementary determiningregion (CDR) 3 having at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%,or preferably 100% sequence identity to an amino acid sequence selectedfrom SEQ ID Nos. 3, 9, 15, 21, 27, 33, 39, 45, 51, 57, 63, 69, 75, 81,129, and 135.

In some embodiments the antigen recognizing construct of the inventionspecifically binds to a TAA-peptide-HLA molecule complex, wherein theTAA peptide comprises, or alternatively consists of, a variant of theTAA which is at least 66%, preferably at least 77%, and more preferablyat least 88% homologous (preferably at least 77% or at least 88%identical) to the amino acid sequence of the TAA of the invention,wherein said variant binds to an HLA class I or class II molecule and/orinduces T-cells cross-reacting with said peptide, or a pharmaceuticallyacceptable salt thereof, wherein said peptide is not the underlyingfull-length polypeptide.

As used herein, the terms “identical” or percent “identity”, when usedanywhere herein in the context of two or more nucleic acid orprotein/polypeptide sequences, refer to two or more sequences orsubsequences that are the same or have (or have at least) a specifiedpercentage of amino acid residues or nucleotides that are the same(i.e., at, or at least, about 60% identity, preferably at, or at least,65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93% or 94%, identity, and morepreferably at, or at least, about 95%, 96%, 97%, 98%, 99%, or higheridentity over a specified region—preferably over their full lengthsequences—, when compared and aligned for maximum correspondence overthe comparison window or designated region) as measured using a sequencecomparison algorithms, or by manual alignment and visual inspection(see, e.g., NCBI web site). In a particular embodiment, for example whencomparing the protein or nucleic acid sequence of an antigen recognizingconstruct of the invention to another protein/gene, the percentageidentity can be determined by the Blast searches supported at the NCBIweb site; in particular for amino acid identity, those using BLASTP withthe following parameters: Expected threshold 10; Word size: 6; Matrix:BLOSUM62; Gap Costs: Existence: 11, Extension: 1; Neighboring wordsthreshold: 11; Compositional adjustments: Conditional compositionalscore matrix adjustment.

In the context of the present invention it shall be understood that anyembodiments referred to as “comprising” certain features of theinvention, shall be understood to include in some more preferredembodiments the more restricted description of “consisting of” or“consisting essentially of” the very same features of the presentinvention.

In another additional or alternative embodiment, the antigen recognizingconstruct may further comprise a CDR1 and/or a CDR2, or more preferablya CDR2bis, domain sequence. Within the variable domain, CDR1 and CDR2 orCDR2bis, are found in the variable (V) region of a polypeptide chain,and CDR3 includes some of V, all of diversity (D) and joining (J)regions. CDR3 is the most variable and is the main CDR responsible forspecifically and selectively recognizing an antigen. CDR1, CDR2 andCDR2bis sequences may be selected from a CDR sequence of a humanvariable chain allele.

Native alpha-beta heterodimeric TCRs have an alpha chain and a betachain. Each chain comprises variable, joining and constant regions, andthe beta chain also usually contains a short diversity region betweenthe variable and joining regions, but this diversity region is oftenconsidered as part of the joining region. Each variable region comprisesthree CDRs (Complementarity Determining Regions) embedded in a frameworksequence, one being the hypervariable region named CDR3. There areseveral types of alpha chain variable (Vα) regions and several types ofbeta chain variable (Vβ) regions distinguished by their framework, CDR1and CDR2 sequences, and by a partly defined CDR3 sequence. The Vα typesare referred to in IMGT nomenclature by a unique TRAV number, Vβ typesare referred to by a unique TRBV number. For more information onimmunoglobulin antibody and TCR genes see the internationalImMunoGeneTics information System®, Lefranc M-P et al (Nucleic AcidsRes. 2015 January; 43(Database issue):D413-22, and imgt.org/).

Therefore, in one additional or alternative embodiment the antigenrecognizing construct of the invention comprises CDR1, CDR2, CDR2bis andCDR3 sequences in a combination as provided in Table 1 herein below,which display the respective variable chain allele together with theCDR3 sequence. Therefore, preferred are antigen recognizing constructsof the invention which comprise at least one, preferably, all four CDRsequences CDR1, CDR2, CDR2bis and CDR3. Preferably, an antigenrecognizing construct of the invention comprises the respective CDR1,CDR2bis and CDR3 of one individual herein disclosed TCR variable regionof the invention (see Table 1 herein below and the example section).

The term “specificity” or “antigen specificity” or “specific for” agiven antigen, as used herein means that the antigen recognizingconstruct can specifically bind to said antigen, preferably a TAAantigen, more preferably with high avidity, when said antigen ispresented by HLA, preferably by HLA A2. For example, a TCR, as antigenrecognizing construct, may be considered to have “antigenic specificity”for the TAA, if T cells expressing the TCR and contacted with a TAApresenting HLA secrete at least about 200 pg/ml or more (e.g., 250 pg/mlor more, 300 pg/ml or more, 400 pg/ml or more, 500 pg/ml or more, 600pg/ml or more, 700 pg/ml or more, 1000 pg ml or more, 2,000 pg/ml ormore, 2,500 pg/ml or more, 5,000 pg/ml or more) of interferon γ (IFN-γ)upon co-culture with target cells pulsed with a low concentration of aTAA antigen, such as the TAA epitopes and antigens provided herein below(e.g., about 10-11 mol/l, 10-10 mol/l, 10-9 mol/l, 10-8 mol/l, 10-7mol/l, 10-6 mol/l, 10-5 mol/l). Alternatively, or additionally, a TCRmay be considered to have “antigenic specificity” for the TAA, if Tcells expressing the TCR secrete at least twice as much IFN-γ as thenon-transduced background level of IFN-γ upon co-culture with targetcells pulsed with a low concentration of the TAA antigens. Such a“specificity” as described above can—for example—be analyzed with anELISA.

In one alternative or additional embodiment of the invention, theantigen recognizing construct selectively binds to a TAA derivedantigenic peptide; preferably wherein the TAA antigenic peptide is aprotein epitope or peptide having an amino acid sequence shown in SEQ IDNO: 97 to 115, most preferably SEQ ID NO:97, or a variant thereof,wherein the variant is an amino acid deletion, addition, insertion orsubstitution of not more than three, preferably two and most preferablynot more than one amino acid position.

The term “selectivity” or “selective recognizing/binding” is understoodto refer to the property of an antigen recognizing construct, such as aTCR or antibody, to selectively recognize or bind to preferably only onespecific epitope and preferably shows no or substantially nocross-reactivity to another epitope. Preferably “selectivity” or“selective recognizing/binding” means that the antigen recognizingconstruct (e.g. a TCR) selectively recognizes or binds to preferablyonly one specific epitope and preferably shows no or substantially nocross-reactivity to another epitope, wherein said epitope is unique forone protein, such that the antigen recognizing construct shows no orsubstantially no cross-reactivity to another epitope and anotherprotein.

The antigen recognizing construct according to the invention ispreferably selected from an antibody, or derivative or fragment thereof,or a T cell receptor (TCR), or derivative or fragment thereof. Aderivative or fragment of an antibody or TCR of the invention shallpreferably retain the antigen binding/recognizing ability of the parentmolecule, in particular its specificity and/or selectivity as explainedabove. Such binding functionality may be retained by the presence of aCDR3 region as defined herein.

In an embodiment of the invention, the inventive TCRs are able torecognize TAA antigens in a major histocompatibility complex (MHC) classI-dependent manner. “MHC class I-dependent manner,” as used herein,means that the TCR elicits an immune response upon binding to TAAantigens within the context of an MHC class I molecule. The MHC class Imolecule can be any MHC class I molecule known in the art, e.g., HLA-Amolecules. In a preferred embodiment of the invention, the MHC class Imolecule is an HLA-A2 molecule.

The invention provides both single chain antigen recognizing constructand double chain recognizing constructs.

In an embodiment, the TCR alpha variable domain has at least onemutation relative to a TCR alpha domain shown in Table 1; and/or the TCRbeta variable domain has at least one mutation relative to a TCR alphadomain shown in Table 1. In an embodiment, a TCR comprising at least onemutation in the TCR alpha variable domain and/or TCR beta variabledomain has a binding affinity for, and/or a binding half-life for, a TAApeptide-HLA molecule complex, which is at least double that of a TCRcomprising the unmutated TCR alpha domain and/or unmutated TCR betavariable domain.

The TCR alpha chains of the present description may further comprise aTCR alpha transmembrane domain and/or a TCR alpha intracellular domain.The TCR beta chains of the present description may further comprise aTCR beta transmembrane domain and/or a TCR beta intracellular domain.

The invention in particular provides a TCR as antigen recognizingconstruct, or fragment or derivative thereof. The TCR preferably is ofhuman, which is understood as being generated from a human TCR locus andtherefore comprising human TCR sequences. Furthermore, the TCR of theinvention may be characterized in that it is of human origin andspecifically recognizes a TAA antigen of the invention.

Another embodiment of the invention additionally or alternativelyprovides the antigen recognizing construct described above, whichinduces an immune response, preferably wherein the immune response ischaracterized by an increase in interferon (IFN) γ levels.

TCRs of the invention may be provided as single chain α or β, or γ andδ, molecules, or alternatively as double chain constructs composed ofboth the α and β chain, or γ and chain.

The antigen recognizing construct of the invention may comprise a TCR αor γ chain; and/or a TCR β or δ chain; wherein the TCR α or γ chaincomprises a CDR3 having at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%,or 100% sequence identity to an amino acid sequence selected from SEQ IDNos. 3, 15, 27, 39, 51, 63, 75, and 129 and/or wherein the TCR β or δchain comprises a CDR3 having at least 50%, 60%, 70%, 80%, 90%, 95%,98%, 99%, or 100% sequence identity to an amino acid sequence selectedfrom SEQ ID Nos. 9, 21, 33, 45, 57, 69, 81, and 135.

Most preferably, in some additional embodiments, wherein the disclosurerefers to antigen recognizing constructs comprising any one, two, threeor all of the CDR1, CDR2, CDR2bis and CDR3 regions of the hereindisclosed TCR chains (see Table 1), such antigen recognizing constructsmay be preferred, which comprise the respective CDR sequence of theinvention with not more than three, two, and preferably only one,modified amino acid residues. A modified amino acid residue may beselected from an amino acid insertion, deletion or substitution. Mostpreferred is that the three, two, preferably only one modified aminoacid residue is the first or last amino acid residue of the respectiveCDR sequence. If the modification is a substitution, then it ispreferable in some embodiments that the substitution is a conservativeamino acid substitution.

If the antigen recognizing construct of the invention is composed of atleast two amino acid chains, such as a double chain TCR, or antigenbinding fragment thereof, the antigen recognizing construct maycomprises in a first polypeptide chain the amino acid sequence accordingto SEQ ID NO: 3, and in a second polypeptide chain the amino acidsequence according to SEQ ID NO: 9; or in a first polypeptide chain theamino acid sequence according to SEQ ID NO: 15, and in a secondpolypeptide chain the amino acid sequence according to SEQ ID NO: 21; orin a first polypeptide chain the amino acid sequence according to SEQ IDNO: 27, and in a second polypeptide chain the amino acid sequenceaccording to SEQ ID NO: 33; or in a first polypeptide chain the aminoacid sequence according to SEQ ID NO: 39, and in a second polypeptidechain the amino acid sequence according to SEQ ID NO: 45; or in a firstpolypeptide chain the amino acid sequence according to SEQ ID NO: 51,and in a second polypeptide chain the amino acid sequence according toSEQ ID NO: 57; or in a first polypeptide chain the amino acid sequenceaccording to SEQ ID NO: 63, and in a second polypeptide chain the aminoacid sequence according to SEQ ID NO: 69; or in a first polypeptidechain the amino acid sequence according to SEQ ID NO: 75, and in asecond polypeptide chain the amino acid sequence according to SEQ ID NO:81; or in a first polypeptide chain the amino acid sequence according toSEQ ID NO: 129, and in a second polypeptide chain the amino acidsequence according to SEQ ID NO: 135. Any one of the aforementioneddouble chain TCR, or antigen binding fragments thereof, are preferredTCR of the present invention. In some embodiments, the CDR3 of thedouble chain TCR of the invention may be mutated. Mutations of the CDR3sequences as provided above preferably include a substitution, deletion,addition, or insertion of not more than three, preferably two, and mostpreferably not more than one amino acid residue. In some embodiments,the first polypeptide chain may be a TCR α or γ chain, and the secondpolypeptide chain may be a TCR β or δ chain. Preferred is thecombination of an αβ or γδ TCR.

The TCR, or the antigen binding fragment thereof, is in some embodimentscomposed of a TCR α and a TCR β chain, or γ and δ chain. Such a doublechain TCR comprises within each chain variable regions, and the variableregions each comprise one CDR1, one CDR2, or more preferably oneCDR2bis, and one CDR3 sequence. The TCRs comprises the CDR1, CDR2,CDR2bis and CDR3 sequences as comprised in the variable chain amino acidsequence of SEQ ID NOs: 4 and 10; or 16 and 22; or 28 and 34; or 40 and46; or 52 and 58; or 64 and 70; or 76 and 82; or 130 and 136.

Some embodiments of the invention pertain to a TCR, or a fragmentthereof, composed of a TCR α and a TCR β chain, wherein said TCRcomprises the variable region sequences having at least 50%, 60%, 70%,80%, 90%, 95%, 98%, 99%, or preferably 100% sequence identity to theamino acid sequence selected from the α and β chain according to SEQ IDNOs: 4 and 10; or 16 and 22; or 28 and 34; or 40 and 46; or 52 and 58;or 64 and 70; or 76 and 82; or 130 and 136.

In a particularly preferred embodiment, the present invention providesan improved TCR, designated as R11P3D3_KE, composed of a TCR α and a TCRβ chain, wherein said TCR comprises the variable region sequences havingat least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or preferably 100%sequence identity to the amino acid sequence selected from the α and βchain according to SEQ ID NOs: 132 and 138. This TCR showed asurprisingly improved functionality in terms of tumor cell recognitionwhen compared to its parent receptor, designated herein as R11P3D3.

The inventive TCRs may further comprise a constant region derived fromany suitable species, such as any mammal, e.g., human, rat, monkey,rabbit, donkey, or mouse. In an embodiment of the invention, theinventive TCRs further comprise a human constant region. In somepreferred embodiments, the constant region of the TCR of the inventionmay be slightly modified, for example, by the introduction ofheterologous sequences, preferably mouse sequences, which may increaseTCR expression and stability. In some preferred embodiments, thevariable region of the TCR of the intervention may be slightly modified,for example, by the introduction of single point mutations to optimizethe TCR stability and/or to enhance TCR chain pairing.

Some embodiments of the invention pertain to a TCR, or a fragmentthereof, composed of a TCR α and a TCR β chain, wherein said TCRcomprises the constant region having at least 50%, 60%, 70%, 80%, 90%,95%, 98%, 99%, or preferably 100% sequence identity to an amino acidsequence selected from of the α and β chain according to SEQ ID NOs: 5and 11; or 17 and 23; or 29 and 35; or 41 and 47; or 53 and 59; or 65and 71; or 77 and 83; or 131 and 137.

The TCR α or γ chain of the invention may further comprise a CDR1 havingat least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100% sequenceidentity to an amino acid sequence selected from SEQ ID Nos. 1, 13, 25,37, 49, 61, 73, and 127; and/or a CDR2 having at least 50%, 60%, 70%,80%, 90%, 95%, 98%, 99%, or 100% sequence identity to an amino acidsequence selected from SEQ ID Nos. 2, 14, 26, 38, 50, 62, 74, and 128;and/or more preferably a CDR2bis having at least 50%, 60%, 70%, 80%,90%, 95%, 98%, 99%, or 100% sequence identity to an amino acid sequenceselected from SEQ ID Nos. 196, 197, 198, 199, 200, 201, 202, and 204.

According to the invention the TCR β or δ chain may further comprise aCDR1 having at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100%sequence identity to an amino acid sequence selected from SEQ ID Nos. 7,19, 31, 43, 55, 67, 79, and 133; and/or a CDR2 having at least 50%, 60%,70%, 80%, 90%, 95%, 98%, 99%, or 100% sequence identity to an amino acidsequence selected from SEQ ID Nos. 8, 20, 32, 44, 56, 68, 80, and 134;and/or more preferably a CDR2bis having at least 50%, 60%, 70%, 80%,90%, 95%, 98%, 99%, or 100% sequence identity to an amino acid sequenceselected from SEQ ID Nos. 8, 20, 32, 44, 56, 68, 80, and 134.

The antigen recognizing construct may in a further embodiment comprise abinding fragment of a TCR, and wherein said binding fragment comprisesin one chain CDR1, CDR2, CDR2bis and CDR3, optionally selected from theCDR1, CDR2, CDR2bis and CDR3 sequences having the amino acid sequencesof SEQ ID Nos. 1, 2, 3, 196; or 7, 8, 9; or 13, 14, 15, 197; or 19, 20,21; or 25, 26, 27, 198; or 31, 32, 33; or 37, 38, 39, 199; or 43, 44,45; or 49, 50, 51, 200; or 55, 56, 57; or 61, 62, 63, 201; or 67, 68,69; or 73, 74, 75, 202; or 79, 80, 81; or 127, 128, 129, 204; or 133,134, 135.

In further embodiments of the invention the antigen recognizingconstruct as described herein elsewhere is a TCR, or a fragment thereof,composed of at least one TCR α and one TCR β chain sequence, whereinsaid TCR α chain sequence comprises the CDR1, CDR2, CDR2bis and CDR3sequences having the amino acid sequences of SEQ ID NO: 1 to 3 and 196,and said TCR β chain sequence comprises the CDR1 to CDR3 sequenceshaving the amino acid sequences of SEQ ID NO: 7 to 9; or wherein saidTCR α chain sequence comprises the CDR1, CDR2, CDR2bis and CDR3sequences having the amino acid sequences of SEQ ID NO: 13 to 15 and197, and said TCR β chain sequence comprises the CDR1 to CDR3 sequenceshaving the amino acid sequences of SEQ ID NO: 19 to 21; or wherein saidTCR α chain sequence comprises the CDR1, CDR2, CDR2bis and CDR3sequences having the amino acid sequences of SEQ ID NO: 25 to 27 and198, and said TCR β chain sequence comprises the CDR1 to CDR3 sequenceshaving the amino acid sequences of SEQ ID NO: 31 to 33; or wherein saidTCR α chain sequence comprises the CDR1, CDR2, CDR2bis and CDR3sequences having the amino acid sequences of SEQ ID NO: 37 to 39 and199, and said TCR β chain sequence comprises the CDR1 to CDR3 sequenceshaving the amino acid sequences of SEQ ID NO: 43 to 45; or wherein saidTCR α chain sequence comprises the CDR1, CDR2, CDR2bis and CDR3sequences having the amino acid sequences of SEQ ID NO: 49 to 51 and200, and said TCR β chain sequence comprises the CDR1 to CDR3 sequenceshaving the amino acid sequences of SEQ ID NO: 55 to 57; or wherein saidTCR α chain sequence comprises the CDR1, CDR2, CDR2bis and CDR3sequences having the amino acid sequences of SEQ ID NO: 61 to 63 and201, and said TCR β chain sequence comprises the CDR1 to CDR3 sequenceshaving the amino acid sequences of SEQ ID NO: 67 to 69; or wherein saidTCR α chain sequence comprises the CDR1, CDR2, CDR2bis and CDR3sequences having the amino acid sequences of SEQ ID NO: 73 to 75 and202, and said TCR β chain sequence comprises the CDR1 to CDR3 sequenceshaving the amino acid sequences of SEQ ID NO: 79 to 81; or wherein saidTCR α chain sequence comprises the CDR1, CDR2, CDR2bis and CDR3sequences having the amino acid sequences of SEQ ID NO: 127 to 129 and204, and said TCR β chain sequence comprises the CDR1 to CDR3 sequenceshaving the amino acid sequences of SEQ ID NO: 133 to 135.

In further embodiments of the invention the antigen recognizingconstruct as described herein before is a TCR, or a fragment thereof,comprising at least one TCR α and one TCR β chain sequence, wherein saidTCR α chain sequence comprises a variable region sequence having theamino acid sequence of SEQ ID No. 4, and wherein said TCR β chainsequence comprises a variable region sequence having the amino acidsequence of SEQ ID No. 10; or wherein said TCR α chain sequencecomprises a variable region sequence having the amino acid sequence ofSEQ ID No. 16, and wherein said TCR β chain sequence comprises avariable region sequence having the amino acid sequence of SEQ ID No.22; or wherein said TCR α chain sequence comprises a variable regionsequence having the amino acid sequence of SEQ ID No. 28, and whereinsaid TCR β chain sequence comprises a variable region sequence havingthe amino acid sequence of SEQ ID No. 34; or wherein said TCR α chainsequence comprises a variable region sequence having the amino acidsequence of SEQ ID No. 40, and wherein said TCR β chain sequencecomprises a variable region sequence having the amino acid sequence ofSEQ ID No. 46; or wherein said TCR α chain sequence comprises a variableregion sequence having the amino acid sequence of SEQ ID No. 52, andwherein said TCR β chain sequence comprises a variable region sequencehaving the amino acid sequence of SEQ ID No. 58; or wherein said TCR αchain sequence comprises a variable region sequence having the aminoacid sequence of SEQ ID No. 64, and wherein said TCR β chain sequencecomprises a variable region sequence having the amino acid sequence ofSEQ ID No. 70; or wherein said TCR α chain sequence comprises a variableregion sequence having the amino acid sequence of SEQ ID No. 76, andwherein said TCR β chain sequence comprises a variable region sequencehaving the amino acid sequence of SEQ ID No. 82; or wherein said TCR αchain sequence comprises a variable region sequence having the aminoacid sequence of SEQ ID No. 130, and wherein said TCR β chain sequencecomprises a variable region sequence having the amino acid sequence ofSEQ ID No. 136.

In further embodiments of the invention the antigen recognizingconstruct as described herein before is a TCR, or a fragment thereof,further comprising a TCR constant region having at least 50%, 60%, 70%,80%, 90%, 95%, 98%, 99%, or 100% sequence identity to an amino acidsequence selected from SEQ ID Nos. 5, 11, 17, 23, 29, 35, 41, 47, 53,59, 65, 71, 77, 83, 131, and 137 preferably wherein the TCR is composedof at least one TCR α and one TCR β chain sequence, wherein the TCR αchain sequence comprises a constant region having at least 50%, 60%,70%, 80%, 90%, 95%, 98%, 99%, or 100% sequence identity to an amino acidsequence selected from SEQ ID Nos. 5, 17, 29, 41, 53, 65, 77, and 131;and wherein the TCR β chain sequence comprises a constant region havingat least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100% sequenceidentity to an amino acid sequence selected from SEQ ID Nos. 11, 23, 35,47, 59, 71, 83, and 137.

Also disclosed are antigen recognizing constructs as described hereinbefore comprising a first TCR chain having at least 50%, 60%, 70%, 80%,90%, 95%, 98%, 99%, or 100% sequence identity to the amino acid sequenceof SEQ ID No. 6, and a second TCR chain having at least 50%, 60%, 70%,80%, 90%, 95%, 98%, 99%, or 100% sequence identity to the amino acidsequence of SEQ ID No. 12. The invention also provides TCRs comprising afirst TCR chain having at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%,or 100% sequence identity to the amino acid sequence of SEQ ID No. 18,and a second TCR chain having at least 50%, 60%, 70%, 80%, 90%, 95%,98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ IDNo. 24. In further embodiments, the invention provides antigenrecognizing constructs which are TCR and comprise a first TCR chainhaving at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100% sequenceidentity to the amino acid sequence of SEQ ID No. 30, and a second TCRchain having at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100%sequence identity to the amino acid sequence of SEQ ID No. 36. Infurther embodiments, the invention provides antigen recognizingconstructs which are TCR and comprise a first TCR chain having at least50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100% sequence identity to theamino acid sequence of SEQ ID No. 42, and a second TCR chain having atleast 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100% sequence identityto the amino acid sequence of SEQ ID No. 48. In further embodiments, theinvention provides antigen recognizing constructs which are TCR andcomprise a first TCR chain having at least 50%, 60%, 70%, 80%, 90%, 95%,98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ IDNo. 54, and a second TCR chain having at least 50%, 60%, 70%, 80%, 90%,95%, 98%, 99%, or 100% sequence identity to the amino acid sequence ofSEQ ID No. 60. In further embodiments, the invention provides antigenrecognizing constructs which are TCR and comprise a first TCR chainhaving at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100% sequenceidentity to the amino acid sequence of SEQ ID No.66, and a second TCRchain having at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100%sequence identity to the amino acid sequence of SEQ ID No. 72. Infurther embodiments, the invention provides antigen recognizingconstructs which are TCR and comprise a first TCR chain having at least50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100% sequence identity to theamino acid sequence of SEQ ID No. 78, and a second TCR chain having atleast 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100% sequence identityto the amino acid sequence of SEQ ID No. 84. In further embodiments, theinvention provides antigen recognizing constructs which are TCR andcomprise a first TCR chain having at least 50%, 60%, 70%, 80%, 90%, 95%,98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ IDNo. 132, and a second TCR chain having at least 50%, 60%, 70%, 80%, 90%,95%, 98%, 99%, or 100% sequence identity to the amino acid sequence ofSEQ ID No. 138.

As used herein, the term “murine” or “human,” when referring to anantigen recognizing construct, or a TCR, or any component of a TCRdescribed herein (e.g., complementarity determining region (CDR),variable region, constant region, α chain, and/or β chain), means a TCR(or component thereof), which is derived from a mouse or a humanunrearranged TCR locus, respectively.

In an embodiment of the invention, chimeric TCR are provided, whereinthe TCR chains comprise sequences from multiple species. Preferably, aTCR of the invention may comprise an α chain comprising a human variableregion of an α chain and, for example, a murine constant region of amurine TCR α chain.

In one embodiment, the TCR of the invention is a human TCR comprisinghuman variable regions according to the above embodiments and humanconstant regions.

In some embodiments, the antigen recognizing construct is murinized orhumanized. These terms are used when amino acid sequences from a foreignspecies are introduced into a construct of the invention.

The TCR of the invention may be provided as a single chain TCR (scTCR).A scTCR according to the invention shall comprise in one polypeptidechain a full or partial alpha chain sequence and a full or partial betachain sequence, preferably connected via a peptide linker. A scTCR cancomprise a polypeptide of a variable region of a first TCR chain (e.g.,an alpha chain) and a polypeptide of an entire (full-length) second TCRchain (e.g., a beta chain), or vice versa. Furthermore, the scTCR canoptionally comprise one or more linkers which join the two or morepolypeptides together. The linker can be, for instance, a peptide, whichjoins together two single chains, as described herein. Also provided issuch a scTCR of the invention, which is fused to a human cytokine, suchas IL-2, IL-7 or IL-15.

The antigen recognizing construct according to the invention can also beprovided in the form of a multimeric complex, comprising at least twoscTCR molecules, wherein said scTCR molecules are each fused to at leastone biotin moiety, or other interconnecting molecule/linker, and whereinsaid scTCRs are interconnected by biotin-streptavidin interaction toallow the formation of said multimeric complex. Similar approaches knownin the art for the generation of multimeric TCR are also possible andincluded in this disclosure. Also provided are multimeric complexes of ahigher order, comprising more than two scTCR of the invention.

For the purposes of the present invention, a TCR is a moiety having atleast one TCR alpha or gamma and/or TCR beta or delta variable domain.Generally, they comprise both a TCR alpha variable domain and a TCR betavariable domain, alternatively both a TCR gamma variable domain and aTCR delta variable domain. They may be αβ/γδ heterodimers or may be insingle chain format. For use in adoptive therapy, an αβ or γδheterodimeric TCR may, for example, be transfected as full-length chainshaving both cytoplasmic and transmembrane domains. If desired, anintroduced disulfide bond between residues of the respective constantdomains may be present.

In a preferred embodiment, the antigen recognizing construct is a humanTCR, or fragment or derivative thereof. A human TCR or fragment orderivative thereof is a TCR, which comprises over 50% of thecorresponding human TCR sequence. Preferably, only a small part of theTCR sequence is of artificial origin or derived from other species. Itis known, however, that chimeric TCRs, e.g. derived from human originwith murine sequences in the constant domains, are advantageous.Particularly preferred are, therefore, TCRs in accordance with thepresent invention, which contains murine sequences in the extracellularpart of their constant domains.

Thus, it is also preferred that the inventive antigen recognizingconstruct is able to recognize its antigen in a human leucocyte antigen(HLA) dependent manner, preferably in a HLA-A*02 dependent manner. Theterm “HLA dependent manner” in the context of the present inventionmeans that the antigen recognizing construct binds to the antigen onlyin the event that the antigenic peptide is presented by said HLA.

The antigen recognizing construct in accordance with the invention inone embodiment preferably induces an immune response, preferably whereinthe immune response is characterized by the increase in interferon (IFN)γ levels.

Also, provided by the invention is a polypeptide comprising a functionalportion of any of the TCRs (or functional variants thereof) describedherein, for examples, of any one of the TCRs selected from R11P3D3,R16P1C10, R16P1E8, R17P1A9, R17P1D7, R17P1G3, R17P2B6 and R11P3D3_KE, asprovided in the example section and Table 1. The term “polypeptide” asused herein includes oligopeptides and refers to a single chain of aminoacids connected by one or more peptide bonds. With respect to theinventive polypeptides, the functional portion can be any portioncomprising contiguous amino acids of the TCR (or functional variantthereof), of which it is a part, provided that the functional portionspecifically binds to the TAA antigen, preferably as disclosed herein inTable 2, and peptides A1 to A9 (SEQ ID NOs:97, and 98-106, and thepeptides T1 to T9 (SEQ ID NOs:107-115)). The term “functional portion”when used in reference to a TCR (or functional variant thereof) refersto any part or fragment of the TCR (or functional variant thereof) ofthe invention, which part or fragment retains the biological activity ofthe TCR (or functional variant thereof), of which it is a part (theparent TCR or parent functional variant thereof). Functional portionsencompass, for example, those parts of a TCR (or functional variantthereof) that retain the ability to specifically bind to the TAA antigen(in an HLA dependent manner), or detect, treat, or prevent cancer, to asimilar extent, the same extent, or to a higher extent, as the parentTCR (or functional variant thereof). In reference to the parent TCR (orfunctional variant thereof), the functional portion can comprise, forinstance, about 10%, 25%, 30%, 50%, 68%, 80%, 90%, 95%, or more, of theparent TCR variable sequences (or functional variant thereof).

The functional portion can comprise additional amino acids at the aminoor carboxy terminus of the portion, or at both termini, in whichadditional amino acids are not found in the amino acid sequence of theparent TCR or functional variant thereof. Desirably, the additionalamino acids do not interfere with the biological function of thefunctional portion, e.g., specifically binding to the TAA antigens;and/or having the ability to detect cancer, treat or prevent cancer,etc. More desirably, the additional amino acids enhance the biologicalactivity, as compared to the biological activity of the parent TCR orfunctional variant thereof.

The polypeptide can comprise a functional portion of either or both ofthe α and β chains of the TCRs or functional variant thereof of theinvention, such as a functional portion comprising one of more of CDR1,CDR2, CDR2bis and (preferably) CDR3 of the variable region(s) of the αchain and/or β chain of a TCR or functional variant thereof of theinvention. In an embodiment of the invention, the polypeptide cancomprise a functional portion comprising the amino acid sequence of SEQID NO: 3, 9, 15, 21, 27, 33, 39, 45, 51, 57, 63, 69, 75, 81, 129, and135 (CDR3 of the variable regions of the TCR of the invention), or acombination thereof. In an embodiment of the invention, the inventivepolypeptide can comprise, for instance, the variable region of theinventive TCR or functional variant thereof comprising a combination ofthe CDR regions set forth above. In this regard, the polypeptide cancomprise the amino acid sequence of any of SEQ ID NO: 4, 10, 16, 22, 28,34, 40, 46, 52, 58, 64, 70, 76, 82, 130, and 136 (the variable regionsof an α or β chain of the TCR of the invention).

In some instances, the construct of the invention may comprise one ortwo polypeptide chains comprising a sequence according to any of the SEQID NO: 1 to 84 and 127 to 138 and 196 to 202 and 204 (CDR sequences,constant and variable regions and full length sequences), or functionalfragments thereof, and further comprise(s) other amino acid sequences,e.g., an amino acid sequence encoding an immunoglobulin or a portionthereof, then the inventive protein can be a fusion protein. In thisregard, the invention also provides a fusion protein comprising at leastone of the inventive polypeptides described herein along with at leastone other polypeptide. The other polypeptide can exist as a separatepolypeptide of the fusion protein, or can exist as a polypeptide, whichis expressed in frame (in tandem) with one of the inventive polypeptidesdescribed herein. The other polypeptide may include any peptidic orproteinaceous molecule, or a portion thereof, including, but not limitedto an immunoglobulin, CD3, CD4, CD8, an MHC molecule, a CD1 molecule,e.g., CD1a, CD1b, CD1c, CD1d, etc.

The fusion protein can comprise one or more copies of the inventivepolypeptide and/or one or more copies of the other polypeptide. Forinstance, the fusion protein can comprise 1, 2, 3, 4, 5, or more, copiesof the inventive polypeptide and/or of the other polypeptide. Suitablemethods of making fusion proteins are known in the art, and include, forexample, recombinant methods. In some embodiments of the invention, theTCRs (and functional portions and functional variants thereof),polypeptides, and proteins of the invention may be expressed as a singleprotein comprising a linker peptide linking the α chain and the β chain,and linking the γ chain and the δ chain. In this regard, the TCRs (andfunctional variants and functional portions thereof), polypeptides, andproteins of the invention comprising the amino acid sequences of thevariable regions of the TCR of the invention and may further comprise alinker peptide. The linker peptide may advantageously facilitate theexpression of a recombinant TCR (including functional portions andfunctional variants thereof), polypeptide, and/or protein in a hostcell. The linker peptide may comprise any suitable amino acid sequence.Linker sequences for single chain TCR constructs are well known in theart. Such a single chain construct may further comprise one, or two,constant domain sequences. Upon expression of the construct includingthe linker peptide by a host cell, the linker peptide may also becleaved, resulting in separated α and β chains, and separated γ and δchain.

As already mentioned above, the binding functionality of the TCR of theinvention may be provided in the framework of an antibody. For example,CDR sequences of the TCR of the invention, possibly including additional3, 2 or 1 N and/or C terminal framework residues, may be directlygrafted into an antibody variable heavy/light chain sequence. The term“antibody” in its various grammatical forms is used herein to refer toimmunoglobulin molecules and immunologically active portions ofimmunoglobulin molecules, i.e., molecules that contain anantigen-binding site or a paratope. Such molecules are also referred toas “antigen binding fragments” of immunoglobulin molecules. Theinvention further provides an antibody, or antigen binding portionthereof, which specifically binds to the antigens described herein. Theantibody can be any type of immunoglobulin that is known in the art. Forinstance, the antibody can be of any isotype, e.g., IgA, IgD, IgE, IgG,IgM, etc. The antibody can be monoclonal or polyclonal. The antibody canbe a naturally-occurring antibody, e.g., an antibody isolated and/orpurified from a mammal, e.g., mouse, rabbit, goat, horse, chicken,hamster, human, etc. Alternatively, the antibody can be agenetically-engineered antibody, e.g., a humanized antibody or achimeric antibody. The antibody can be in monomeric or polymeric form.

The term “antibody” includes, but is not limited to, geneticallyengineered or otherwise modified forms of immunoglobulins, such asintrabodies, chimeric antibodies, fully human antibodies, humanizedantibodies (e.g. generated by “CDR-grafting”), antibody fragments, andheteroconjugate antibodies (e.g., bispecific antibodies, diabodies,triabodies, tetra-bodies, etc.). The term “antibody” includescys-diabodies and minibodies. Thus, each and every embodiment providedherein in regard to “antibodies”, or “antibody like constructs” is alsoenvisioned as, bi-specific antibodies, diabodies, scFv fragments,chimeric antibody receptor (CAR) constructs, diabody and/or minibodyembodiments, unless explicitly denoted otherwise. The term “antibody”includes a polypeptide of the immunoglobulin family or a polypeptidecomprising fragments of an immunoglobulin that is capable ofnon-covalently, reversibly, and in a specific manner binding acorresponding antigen, preferably the TAA of the invention, as disclosedherein. An exemplary antibody structural unit comprises a tetramer. Insome embodiments, a full-length antibody can be composed of twoidentical pairs of polypeptide chains, each pair having one “light” andone “heavy” chain (connected through a disulfide bond). Antibodystructure and isotypes are well known to the skilled artisan (forexample from Janeway's Immunobiology, 9th edition, 2016).

The recognized immunoglobulin genes of mammals include the kappa,lambda, alpha, gamma, delta, epsilon, and mu constant region genes, aswell as the myriad immunoglobulin variable region genes (for moreinformation on immunoglobulin genes see the internationalIm-MunoGeneTics information System®, Lefranc M-P et al, Nucleic AcidsRes. 2015 January; 43(Database issue):D413-22; and imgt.org/). Forfull-length chains, the light chains are classified as either kappa orlambda. For full-length chains, the heavy chains are classified asgamma, mu, alpha, delta, or epsilon, which in turn define theimmunoglobulin classes, IgG, IgM, IgA, IgD, and IgE, respectively. TheN-terminus of each chain defines a variable region of about 100 to 110or more amino acids primarily responsible for antigen recognition. Theterms variable light chain (VL) and variable heavy chain (VH) refer tothese regions of light and heavy chains respectively. As used in thisinvention, an “antibody” encompasses all variations of antibody andfragments thereof. Thus, within the scope of this concept are fulllength antibodies, chimeric antibodies, humanized antibodies, singlechain antibodies (scFv), Fab, Fab′, and multimeric versions of thesefragments (e.g., F(ab′)2) with the same, essentially the same or similarbinding specificity. In some embodiments, the anti-body bindsspecifically to a peptide TAA of the invention. Preferred antigenrecognizing constructs according to the invention include an antibodyheavy chain, preferably the variable domain thereof, or an antigenbinding fragment thereof, and/or an antibody light chain, preferably thevariable domain thereof, or an antigen binding fragment thereof.Similarly, disulfide-stabilized variable region fragments (dsFv) can beprepared by recombinant DNA technology, antibody fragments of theinvention, however, are not limited to these exemplary types of antibodyfragments. Also, the antibody, or antigen binding portion thereof, canbe modified to comprise a detectable label, such as, for instance, aradioisotope, a fluorophore (e.g., fluorescein isothiocyanate (FITC),phycoerythrin (PE)), an enzyme (e.g., alkaline phosphatase, horseradishperoxidase), and element particles (e.g., gold particles). In someinstances, the TCR CDR3 sequence may be slightly modified, butpreferably by not more than 3 amino acid residues, preferably only twoand most preferably only one amino acid position, as compared to theCDR3 sequences provided in SEQ ID Nos: 3, 9, 15, 21, 27, 33, 39, 45, 51,57, 63, 69, 75, 81, 129, and 135. Preferably, the antibodies comprisethe CDR3, preferably all of CDR1, CDR2, CDR2bis and CDR3 regions in thecombination, as indicated for the TCR of the invention in Table 1, ineach case independently, optionally with not more than three or two,preferably one, amino acid substitution(s), insertion(s) and/ordeletion(s) compared to these sequences.

Suitable methods of making antibodies are known in the art. Forinstance, standard hybridoma methods are described in, e.g., Kohler andMilstein, Eur. J. Immunol, 5, 51 1-519 (1976), Harlow and Lane (eds.),Antibodies: A Laboratory Manual, CSH Press (1988), and C. A. Janeway etal. (eds.), Immunobiology, 8 Ed., Garland Publishing, New York, N.Y.(2011)). Alternatively, other methods, such as EBV-hybridoma methods(Haskard and Archer, J. Immunol. Methods, 74(2), 361-67 (1984), andRoder et al, Methods Enzymol, 121, 140-67 (1986)), and bacteriophagevector expression systems (see, e.g., Huse et al., Science, 246, 1275-81(1989)) are known in the art. Further, methods of producing antibodiesin non-human animals are described in, e.g., U.S. Pat. Nos. 5,545,806,5,569,825, and 5,714,352, and U.S. Patent Application Publication No.2002/0197266.

Some embodiments of the invention also pertain to TCRs, or functionalfragments and polypeptides thereof, which are soluble TCRs. As usedherein, the term “soluble T-cell receptor” refers to heterodimerictruncated variants of native TCRs, which comprise extracellular portionsof the TCR α-chain and β-chain, for example linked by a disulfide bond,but which lack the transmembrane and cytosolic domains of the nativeprotein. The terms “soluble T-cell receptor α-chain sequence and solubleT-cell receptor β-chain sequence” refer to TCR α-chain and β-chainsequences that lack the transmembrane and cytosolic domains. Thesequence (amino acid or nucleic acid) of the soluble TCR α-chain andβ-chains may be identical to the corresponding sequences in a native TCRor may comprise variant soluble TCR α-chain and β-chain sequences, ascompared to the corresponding native TCR sequences. The term “solubleT-cell receptor” as used herein encompasses soluble TCRs with variant ornon-variant soluble TCR α-chain and β-chain sequences. The variationsmay be in the variable or constant regions of the soluble TCR α-chainand β-chain sequences and can include, but are not limited to, aminoacid deletion, insertion, substitution mutations as well as changes tothe nucleic acid sequence, which do not alter the amino acid sequence.Soluble TCR of the invention in any case retain the bindingfunctionality of their parent molecules.

The above problem is further solved by a nucleic acid encoding for anantigen recognizing construct of the invention, or any of theaforementioned protein or polypeptide constructs. The nucleic acidpreferably (a) has a strand encoding for an antigen recognizingconstruct according to the invention; (b) has a strand complementary tothe strand in (a); or (c) has a strand that hybridizes under stringentconditions with a molecule as described in (a) or (b). Stringentconditions are known to the person of skill in the art, specificallyfrom Sambrook et al, “Molecular Cloning”. In addition to that, thenucleic acid optionally has further sequences, which are necessary forexpressing the nucleic acid sequence corresponding to the protein,specifically for expression in a mammalian/human cell. The nucleic acidused can be contained in a vector suitable for allowing expression ofthe nucleic acid sequence corresponding to the peptide in a cell.However, the nucleic acids can also be used to transform anantigen-presenting cell, which may not be restricted to classicalantigen-presenting cells, such as dendritic cells, in such a way thatthey themselves produce the corresponding proteins on their cellularsurface.

In some embodiments, the polypeptides of the antigen recognizingconstructs can be encoded by nucleic acids and expressed in vivo or invitro. Thus, in some embodiments, a nucleic acid encoding an antigenrecognizing construct is provided. In some embodiments, the nucleic acidencodes one part or monomer of an antigen recognizing construct of theinvention (for example one of two chains of a TCR of the invention),and/or another nucleic acid encodes another part or monomer of anantigen recognizing construct of the invention (for example the other oftwo chains of the TCR). In some embodiments, the nucleic acid encodestwo or more antigens recognizing construct polypeptide chains, forexample, at least 2 TCR chains. Nucleic acids encoding multiple antigenrecognizing construct chains can include nucleic acid cleavage sitesbetween at least two chain sequences, can encode transcription ortranslation start site between two or more chains sequences, and/or canencode proteolytic target sites between two or more antigen recognizingconstruct chains.

By “nucleic acid” as used herein includes “polynucleotide,”“oligonucleotide,” and “nucleic acid molecule,” and generally means apolymer of DNA or RNA, which can be single-stranded or double-stranded,synthesized or obtained (e.g., isolated and/or purified) from naturalsources, which can contain natural, non-natural or altered nucleotides,and can contain a natural, non-natural or altered internucleotidelinkage, such as a phosphoroamidate linkage or a phosphorothioatelinkage, instead of the phosphodiester found between the nucleotides ofan unmodified oligonucleotide.

Preferably, the nucleic acids of the invention are recombinant. As usedherein, the term “recombinant” refers to (i) molecules that areconstructed outside living cells by joining natural or synthetic nucleicacid segments to nucleic acid molecules that can replicate in a livingcell, or (ii) molecules that result from the replication of thosedescribed in (i) above. For purposes, herein, the replication can be invitro replication or in vivo replication. The nucleic acid can compriseany nucleotide sequence, which encodes any of the TCRs, polypeptides, orproteins, or functional portions or functional variants thereofdescribed herein.

Furthermore, the invention provides a vector comprising a nucleic acidin accordance to the invention as described above. Desirably, the vectoris an expression vector or a recombinant expression vector. The term“recombinant expression vector” refers in context of the presentinvention to a nucleic acid construct that allows for the expression ofan mRNA, protein or polypeptide in a suitable host cell. The recombinantexpression vector of the invention can be any suitable recombinantexpression vector, and can be used to transform or transfect anysuitable host. Suitable vectors include those designed for propagationand expansion or for expression or both, such as plasmids and viruses.Examples of animal expression vectors include pEUK-CI, pMAM, andpMAMneo. Preferably, the recombinant expression vector is a viralvector, e.g., a retroviral vector. The recombinant expression vectorcomprises regulatory sequences, such as transcription and translationinitiation and termination codons, which are specific to the type ofhost cell (e.g., bacterium, fungus, plant, or animal), into which thevector is to be introduced and in which the expression of the nucleicacid of the invention may be performed. Furthermore, the vector of theinvention may include one or more marker genes, which allow forselection of transformed or transfected hosts. The recombinantexpression vector can comprise a native or normative promoter operablylinked to the nucleotide sequence encoding the constructs of theinvention, or to the nucleotide sequence, which is complementary to orwhich hybridizes to the nucleotide sequence encoding the constructs ofthe invention. The selections of promoters include, e.g., strong, weak,inducible, tissue-specific and developmental-specific promoters. Thepromoter can be a non-viral promoter or a viral promoter. The inventiverecombinant expression vectors can be designed for either transientexpression, for stable expression, or for both. Also, the recombinantexpression vectors can be made for constitutive expression or forinducible expression.

The invention also pertains to a host cell comprising an antigenrecognizing construct in accordance with the invention. Specifically,the host cell of the invention comprises a nucleic acid, or a vector asdescribed herein above. The host cell can be a eukaryotic cell, e.g.,plant, animal, fungi, or algae, or can be a prokaryotic cell, e.g.,bacteria or protozoa. The host cell can be a cultured cell or a primarycell, i.e., isolated directly from an organism, e.g., a human. The hostcell can be an adherent cell or a suspended cell, i.e., a cell thatgrows in suspension. For purposes of producing a recombinant TCR,polypeptide, or protein, the host cell is preferably a mammalian cell.Most preferably, the host cell is a human cell. While the host cell canbe of any cell type, can originate from any type of tissue, and can beof any developmental stage, the host cell preferably is a peripheralblood leukocyte (PBL) or a peripheral blood mononuclear cell (PBMC).More preferably, the host cell is a T cell. The T cell can be any Tcell, such as a cultured T cell, e.g., a primary T cell, or a T cellfrom a cultured T cell line, e.g., Jurkat, SupT1, etc., or a T cellobtained from a mammal, preferably a T cell or T cell precursor from ahuman patient. If obtained from a mammal, the T cell can be obtainedfrom numerous sources, including but not limited to blood, bone marrow,lymph node, the thymus, or other tissues or fluids. T cells can also beenriched for or purified.

Preferably, the T cell is a human T cell. More preferably, the T cell isa T cell isolated from a human. The T cell can be any type of T cell andcan be of any developmental stage, including but not limited to,CD4-positive and/or CD8-positive, CD4-positive helper T cells, e.g., Th1and Th2 cells, CD8-positive T cells (e.g., cytotoxic T cells), tumorinfiltrating cells (TILs), memory T cells, naive T cells, and the like.Preferably, the T cell is a CD8-positive T cell or a CD4-positive Tcell.

Preferably, the host cell of the invention is a lymphocyte, preferably,a T lymphocyte, such as a CD4-positive or CD8-positive T-cell. The hostcell furthermore preferably is a tumor reactive T cell specific for TAAexpressing tumor cells.

The objective of the invention is also solved by a method ofmanufacturing a TAA specific antigen recognizing construct, or of a TAAspecific antigen recognizing construct expressing cell line, comprising

-   -   a. Providing a suitable host cell,    -   b. Providing a genetic construct comprising a coding sequence        encoding for an antigen recognizing construct according to the        herein disclosed invention,    -   c. Introducing into said suitable host cell said genetic        construct, and    -   d. Expressing said genetic construct by said suitable host cell.

The method may further comprise a step of cell surface presentation ofsaid antigen recognizing construct on said suitable host cell.

In other preferred embodiments, the genetic construct is an expressionconstruct comprising a promoter sequence operably linked to said codingsequence. Preferably, said antigen recognizing construct is of mammalianorigin, preferably of human origin. The preferred suitable host cell foruse in the method of the invention is a mammalian cell, such as a humancell, in particular a human T lymphocyte. T cells for use in theinvention are described in detail herein above.

Also, encompassed by the invention are embodiments, wherein said antigenrecognizing construct is a modified TCR, wherein said modification isthe addition of functional domains, such as a label or a therapeuticallyactive substance. Furthermore, encompassed are TCR having alternativedomains, such as an alternative membrane anchor domain instead of theendogenous transmembrane region. Also, encompassed are TCR having pointmutations in the TCR variable domain or constant domain in order toimprove TCR expression or stability and/or chain pairing.

Desirably, the transfection system for introducing the genetic constructinto said suitable host cell is a retroviral vector system. Such systemsare well known to the skilled artisan.

Also, comprised by the present invention is in one embodiment theadditional method step of isolation and purification of the antigenrecognizing construct from the cell and, optionally, the reconstitutionof the translated antigen recognizing construct-fragments in a T-cell.

In an alternative aspect of the invention a T-cell is provided obtainedor obtainable by a method for the production of a T cell receptor (TCR),which is specific for tumorous cells and has high avidity as describedherein above. Such a T cell is depending on the host cell used in themethod of the invention, for example, a human or non-human T-cell,preferably a human TCR.

The term “isolated” as used herein in the context of a polypeptide, suchas an antigen recognizing construct (an example of which could be anantibody), refers to a polypeptide that is purified from proteins orpolypeptides or other contaminants that would interfere with itstherapeutic, diagnostic, prophylactic, research or other use. An antigenrecognizing construct according to the invention may be a recombinant,synthetic or modified (non-natural) antigen binding construct. The term“isolated” as used herein in the context of a nucleic acid or cellsrefers to a nucleic acid or cells that is/are purified from DNA, RNA,proteins or polypeptides or other contaminants (such as other cells)that would interfere with its therapeutic, diagnostic, prophylactic,research or other use, or it refers to a recombinant, synthetic ormodified (non-natural) nucleic acid. In this context, a “recombinant”protein/polypeptide or nucleic acid is one made using recombinanttechniques. Methods and techniques for the production of recombinantnucleic acids and proteins are well known in the art.

One additional aspect of the present invention relates to the hereindisclosed antigen recognizing constructs, nucleic acids, vectors,pharmaceutical compositions and/or host cell for use in medicine. Theuse in medicine in one preferred embodiment includes the use in thediagnosis, prevention and/or treatment of a tumor disease, such as amalignant or benign tumor disease. The tumor disease is, for example, atumor disease characterized by the expression of the TAA, in a cancer ortumor cell of said tumor disease.

With respect to the above mentioned medical applications of the antigenrecognizing constructs and other materials derived therefrom, pertainingthereto or encoding the same, in accordance of the present disclosure,the to be treated and/or to be diagnosed diseases can be anyproliferative disorder, preferably characterized by the expression ofthe TAA or TAA epitope sequence of the invention, for example anycancer, including any of acute lymphocytic cancer, acute myeloidleukemia, alveolar rhabdomyosarcoma, bone cancer, brain cancer, breastcancer, cancer of the anus, anal canal, or anorectum, cancer of the eye,cancer of the intrahepatic bile duct, cancer of the joints, cancer ofthe neck, gallbladder, or pleura, cancer of the nose, nasal cavity, ormiddle ear, cancer of the oral cavity, cancer of the vagina, cancer ofthe vulva, chronic lymphocytic leukemia, chronic myeloid cancer, coloncancer, esophageal cancer, cervical cancer, gastrointestinal carcinoidtumor, glioma, Hodgkin lymphoma, hypopharynx cancer, kidney cancer,larynx cancer, liver cancer, lung cancer, malignant mesothelioma,melanoma, multiple myeloma, nasopharynx cancer, non-Hodgkin lymphoma,cancer of the oropharynx, ovarian cancer, cancer of the penis,pancreatic cancer, peritoneum, omentum, and mesentery cancer, pharynxcancer, prostate cancer, rectal cancer, renal cancer, skin cancer, smallintestine cancer, soft tissue cancer, stomach cancer, testicular cancer,thyroid cancer, cancer of the uterus, ureter cancer, and urinary bladdercancer. A preferred cancer is cancer of the uterine cervix, oropharynx,anus, anal canal, anorectum, vagina, vulva, or penis. A particularlypreferred cancer is a TAA positive cancer, including preferably ovariancarcinoma, leukemia or melanoma.

The constructs, proteins, TCRs antibodies, polypeptides and nucleicacids of the invention are in particular for use in immune therapy,preferably, in adoptive T cell therapy. The administration of thecompounds of the invention can, for example, involve the infusion of Tcells of the invention into said patient. Preferably, such T cells areautologous T cells of the patient and in vitro transduced with a nucleicacid or antigen recognizing construct of the present invention.

The inventive antigen recognizing constructs, TCRs, polypeptides,proteins (including functional variants thereof), nucleic acids,recombinant expression vectors, host cells (including populationsthereof), and antibodies (including antigen binding portions thereof),all of which are collectively referred to as “inventive TCR materials”hereinafter, can be formulated into a composition, such as apharmaceutical composition. In this regard, the invention provides apharmaceutical composition comprising any of the antigen recognizingconstructs, TCRs, polypeptides, proteins, functional portions,functional variants, nucleic acids, expression vectors, host cells(including populations thereof), and antibodies (including antigenbinding portions thereof) described herein, and a pharmaceuticallyacceptable carrier, excipient and/or stabilizer. The inventivepharmaceutical compositions containing any of the inventive TCRmaterials can comprise more than one inventive TCR material, e.g., apolypeptide and a nucleic acid, or two or more different TCRs (includingfunctional portions and functional variants thereof). Alternatively, thepharmaceutical composition can comprise an inventive TCR material incombination with another pharmaceutically active agent(s) or drug(s),such as chemotherapeutic agents, e.g., asparaginase, busulfan,carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil,gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab,vinblastine, vincristine, etc. Preferably, the carrier is apharmaceutically acceptable carrier. With respect to pharmaceuticalcompositions, the carrier can be any of those conventionally used forthe particular inventive TCR material under consideration. Suchpharmaceutically acceptable carriers are well-known to those skilled inthe art and are readily available to the public. It is preferred thatthe pharmaceutically acceptable carrier be one, which has no detrimentalside effects or toxicity under the conditions of use.

Thus, also provided is a pharmaceutical composition, comprising any ofthe herein described products of the invention and TCR materials of theinvention, specifically any proteins, nucleic acids or host cells. In apreferred embodiment, the pharmaceutical composition is for immunetherapy, preferably adoptive cell therapy.

Preferably, the inventive TCR material is administered by injection,e.g., intravenously.

When the inventive TCR material is a host cell expressing the inventiveTCR (or functional variant thereof), the pharmaceutically acceptablecarrier for the cells for injection may include any isotonic carriersuch as, for example, normal saline (about 0.90% w/v of NaCl in water,about 300 mOsm/L NaCl in water, or about 9.0 g NaCl per liter of water),NORMOSOL R electrolyte solution (Abbott, Chicago, Ill.), PLASMA-LYTE A(Baxter, Deerfield, Ill.), about 5% dextrose in water, or Ringer'slactate. In an embodiment, the pharmaceutically acceptable carrier issupplemented with human serum albumen.

For purposes of the invention, the amount or dose (e.g., numbers ofcells when the inventive TCR material is one or more cells) of theinventive TCR material administered may be sufficient to affect, e.g., atherapeutic or prophylactic response, in the subject or animal over areasonable time frame. For example, the dose of the inventive TCRmaterial should be sufficient to bind to a cancer antigen, or detect,treat or prevent cancer in a period of from about 2 hours or longer,e.g., 12 to 24 or more hours, from the time of administration. Incertain embodiments, the time period could be even longer. The dose willbe determined by the efficacy of the particular inventive TCR materialand the condition of the animal (e.g., human), as well as the bodyweight of the animal (e.g., human) to be treated.

It is contemplated that the inventive pharmaceutical compositions,antigen recognizing constructs, TCRs (including functional variantsthereof), polypeptides, proteins, nucleic acids, recombinant expressionvectors, host cells, or populations of cells can be used in methods oftreating or preventing cancer, or TAA-positive premalignancy. Theinventive TCRs (and functional variants thereof) are believed to bindspecifically to the TAA of the invention, such that the TCR (or relatedinventive polypeptide or protein and functional variants thereof), whenexpressed by or on a cell, such as a T cell, is able to mediate animmune response against a target cell expressing the TAA of theinvention, preferably presenting TAA peptides via MHC I or II on thesurface of said target cell. In this regard, the invention provides amethod of treating or preventing a condition, in particular cancer, in amammal, comprising administering to the mammal any of the pharmaceuticalcompositions, antigen recognizing constructs, in particular TCRs (andfunctional variants thereof), polypeptides, or proteins describedherein, any nucleic acid or recombinant expression vector comprising anucleotide sequence encoding any of the TCRs (and functional variantsthereof), polypeptides, proteins described herein, or any host cell orpopulation of cells comprising a nucleic acid or recombinant vector,which encodes any of the constructs of the invention (and functionalvariants thereof), polypeptides, or proteins described herein, in anamount effective to treat or prevent the condition in the mammal,wherein the condition is preferably cancer, such as a cancer expressingthe TAA of the invention.

Examples of pharmaceutically acceptable carriers or diluents useful inthe present invention include stabilizers such as SPGA, carbohydrates(e.g. sorbitol, mannitol, starch, sucrose, glucose, dextran), proteinssuch as albumin or casein, protein containing agents such as bovineserum or skimmed milk and buffers (e.g. phosphate buffer).

The terms “treat,” and “prevent” as well as words stemming therefrom, asused herein, do not necessarily imply 100% or complete treatment orprevention. Rather, there are varying degrees of treatment or preventionof which one of ordinary skill in the art recognizes as having apotential benefit or therapeutic effect. In this respect, the inventivemethods can provide any amount of any level of treatment or preventionof a condition in a mammal. Furthermore, the treatment or preventionprovided by the inventive method can include treatment or prevention ofone or more conditions or symptoms of the condition, e.g., cancer, beingtreated or prevented. For example, treatment or prevention can includepromoting the regression of a tumor. Also, for purposes herein,“prevention” can encompass delaying the onset of the condition, or asymptom or condition thereof.

The present invention also relates to a method of treating cancercomprising administering a TCR, a nucleic acid, or a host cell of thepresent description in combination with at least one chemotherapeuticagent and/or radiation therapy.

Another aspect of the invention further pertains to a method fordetecting a TAA protein, or a complex of MHC and the TAA protein(protein epitope of the TAA), in a (biological) sample—such as oneobtained from a subject or patient comprising contacting the sample withan antigen recognizing construct specifically binding to said TAApeptide, or to the TAA peptide/MHC complex, and detecting the bindingbetween said antigen recognizing construct and said TAA peptide, or tothe TAA peptide/MHC complex. In some embodiments, the antigenrecognizing construct is a TCR or antibody, or similar constructs, orpreferably the antigen recognizing construct according to the hereindescribed invention. In some embodiments, the (biological) sample is asample of a tumor or a cancer (such as one of those described elsewhereherein) for example a sample comprising tumor or cancer cells.

Also provided is a method of treating cancer in a subject in needthereof, comprising:

a) isolating a cell from said subject;

b) transforming the cell with at least one vector encoding an antigenrecognizing construct of the present invention to produce a transformedcell;

c) expanding the transformed cell to produce a plurality of transformedcells; and

d) administering the plurality of transformed cells to said subject.

Also provided is a method of treating cancer in a subject in needthereof, comprising:

a) isolating a cell from a healthy donor;

b) transforming the cell with a vector encoding an antigen recognizingconstruct of the present invention to produce a transformed cell;

c) expanding the transformed cell to produce a plurality of transformedcells; and

d) administering the plurality of transformed cells to said subject.

Also provided is a method of detecting cancer in a biological samplecomprising:

a) contacting the biological sample with an antigen recognizingconstruct of the present description;

b) detecting binding of the antigen recognizing construct to thebiological sample.

In some embodiments, the method of detecting cancer is carried out invitro, in vivo or in situ.

Also provided is a method of detecting the presence of a condition in amammal. The method comprises (i) contacting a sample comprising one ormore cells from the mammal with any of the inventive TCRs (andfunctional variants thereof), polypeptides, proteins, nucleic acids,recombinant expression vectors, host cells, populations of cells,antibodies, or antigen binding portions thereof, or pharmaceuticalcompositions described herein, thereby forming a complex, and detectingthe complex, wherein detection of the complex is indicative of thepresence of the condition in the mammal, wherein the condition iscancer, such as a TAA expressing malignancy.

With respect to the inventive method of detecting a condition in amammal, the sample of cells can be a sample comprising whole cells,lysates thereof, or a fraction of the whole cell lysates, e.g., anuclear or cytoplasmic fraction, a whole protein fraction, or a nucleicacid fraction.

For purposes of the inventive detection method, the contacting can takeplace in vitro or in vivo with respect to the mammal. Preferably, thecontacting is in vitro.

Also, detection of the complex can occur through any number of waysknown in the art. For instance, the inventive antigen recognizingconstructs (and functional variants thereof), polypeptides, proteins,nucleic acids, recombinant expression vectors, host cells, populationsof cells, or antibodies or TCRs, or antigen binding portions thereof,described herein, can be labeled with a detectable label such as, forinstance, a radioisotope, a fluorophore (e.g., fluoresceinisothiocyanate (FITC), phycoerythrin (PE)), an enzyme (e.g., alkalinephosphatase, horseradish peroxidase), and element particles (e.g., goldparticles).

For purposes of the inventive methods, wherein host cells or populationsof cells are administered, the cells can be cells that are allogeneic orautologous to the mammal. Preferably, the cells are autologous to themammal.

With respect to the above mentioned medical applications of the TCRmaterial of the invention, the to be treated and/or diagnosed cancer canbe any cancer, including any of acute lymphocytic cancer, acute myeloidleukemia, alveolar rhabdomyosarcoma, bone cancer, brain cancer, breastcancer, cancer of the anus, anal canal, or anorectum, cancer of the eye,cancer of the intrahepatic bile duct, cancer of the joints, cancer ofthe neck, gallbladder, or pleura, cancer of the nose, nasal cavity, ormiddle ear, cancer of the oral cavity, cancer of the vagina, cancer ofthe vulva, chronic lymphocytic leukemia, chronic myeloid cancer, coloncancer, esophageal cancer, cervical cancer, gastrointestinal carcinoidtumor, glioma, Hodgkin lymphoma, hypopharynx cancer, kidney cancer,larynx cancer, liver cancer, lung cancer, malignant mesothelioma,melanoma, multiple myeloma, nasopharynx cancer, non-Hodgkin lymphoma,cancer of the oropharynx, ovarian cancer, cancer of the penis,pancreatic cancer, peritoneum, omentum, and mesentery cancer, pharynxcancer, prostate cancer, rectal cancer, renal cancer, skin cancer, smallintestine cancer, soft tissue cancer, stomach cancer, testicular cancer,thyroid cancer, cancer of the uterus, ureter cancer, and urinary bladdercancer. A preferred cancer is cancer is cancer of the uterine cervix,oropharynx, anus, anal canal, anorectum, vagina, vulva, or penis. Aparticularly preferred cancer is a TAA positive cancer, such as a PRAMEexpressing cancer, for example ovarian carcinoma, melanoma or leukemia.

In general, the invention provides a method for treating a subjectsuffering from a tumor or tumor disease comprising the administration ofthe antigen recognizing constructs, nucleic acids, vectors,pharmaceutical compositions and/or host cell as disclosed by the presentinvention. Preferably the subject is a subject in need of such atreatment. The subject in preferred embodiments is a mammalian subject,preferably a human patient, suffering from a tumor or tumor disease,which is TAA-positive.

In view of the disclosure herein it will be appreciated that theinvention furthermore pertains to the following items:

Item 1: An antigen recognizing construct comprising at least onecomplementary determining region (CDR) 3 having at least 50% sequenceidentity to an amino acid sequence selected from SEQ ID NOs. 3, 9, 15,21, 27, 33, 39, 45, 51, 57, 63, 69, 75, 81, 129, and 135.

Item 2: The antigen recognizing construct according to item 1, whereinsaid antigen recognizing construct is capable of specifically and/orselectively binding to a TAA of the invention antigenic peptide.

Item 3: The antigen recognizing construct according to item 1 or 2,wherein the antigen recognizing construct is an antibody, or derivativeor fragment thereof, or a T cell receptor (TCR), or a derivative orfragment thereof.

Item 4: The antigen recognizing construct according to any one of items1 to 3, wherein said antigen recognizing construct binds to a humanleucocyte antigen (HLA) presented TAA antigenic peptide, wherein saidHLA is optionally type A2.

Item 5: The antigen recognizing construct according to any one of items1 to 4, wherein the construct specifically and/or selectively binds toan epitope having the amino acid sequence selected from SEQ ID NO:97-115, preferably SEQ ID NO: 97.

Item 6: The antigen recognizing construct according to any one of items1 to 5, wherein the construct is an α/β-TCR, or fragment or derivativethereof, or the construct is a γ/δ-TCR, or a fragment or derivativethereof.

Item 7: The antigen recognizing construct according to any one of items1 to 6, characterized in that the construct is of human origin andspecifically and/or selectively recognizes a TAA antigenic peptide.

Item 8: The antigen recognizing construct according to any one of items1 to 7, wherein said antigen recognizing construct is capable ofinducing an immune response in a subject, optionally wherein the immuneresponse is characterized by an increase in interferon (IFN) γ levels.

Item 9: The antigen recognizing construct according to any one of items1 to 8, comprising a TCR α or γ chain; and/or a TCR β or δ chain;wherein the TCR α or γ chain comprises a CDR3 having at least 50%, 60%,70%, 80%, 90%, 95%, 98%, 99%, or 100% sequence identity to an amino acidsequence selected from SEQ ID Nos. 3, 15, 27, 39, 51, 63, 75, and 129,and/or wherein the TCR β or δ chain comprises a CDR3 having at least50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100% sequence identity to anamino acid sequence selected from SEQ ID Nos. 9, 21, 33, 45, 57, 69, 81,and 135.

Item 10: The antigen recognizing construct according to item 9, whereinthe TCR α or γ chain further comprises a CDR1 having at least 50%, 60%,70%, 80%, 90%, 95%, 98%, 99%, or 100% sequence identity to an amino acidsequence selected from SEQ ID Nos. 1, 13, 25, 37, 49, 61, 73, and 127;and/or a CDR2 having at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or100% sequence identity to an amino acid sequence selected from SEQ IDNos. 2, 14, 26, 38, 50, 62, 74, 128, 196, 197, 198, 199, 200, 201, 202,and 204.

Item 11: The antigen recognizing construct according to item 9 or 10,wherein the TCR β or δ chain further comprises a CDR1 having at least50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100% sequence identity to anamino acid sequence selected from SEQ ID Nos. 7, 19, 31, 43, 55, 67, and79, and 133; and/or a CDR2 having at least 50%, 60%, 70%, 80%, 90%, 95%,98%, 99%, or 100% sequence identity to an amino acid sequence selectedfrom SEQ ID Nos. 8, 20, 32, 44, 56, 68, and 80, and 134.

Item 12: The antigen recognizing construct according to any one of items1 to 11, comprising a TCR variable chain region having at least 50%,60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100% sequence identity to an aminoacid sequence selected from SEQ ID Nos. 4, 10, 16, 22, 28, 34, 40, 46,52, 58, 64, 70, 76, 82, 130, and 136.

Item 13: The antigen recognizing construct according to any one of items1 to 12, wherein the construct is humanized, chimerized and/ormurinized.

Item 14: The antigen recognizing construct according to any one of items1 to 13, comprising a binding fragment of a TCR, and wherein saidbinding fragment comprises CDR1 to CDR3 optionally selected from theCDR1 to CDR3 sequences having the amino acid sequences of SEQ ID Nos. 1,2, 3, 196; or 7, 8, 9; or 13, 14, 15, 197; or 19, 20, 21; or 25, 26, 27,198; or 31, 32, 33; or 37, 38, 39, 199; or 43, 44, 45; or 49, 50, 51,200; or 55, 56, 57; or 61, 62, 63, 201; or 67, 68, 69; or 73, 74, 75,202; or 79, 80, 81; or 127, 128, 129, 204; or 133, 134, 135.

Item 15: The antigen recognizing construct according to any one of items1 to 14, wherein the construct is a TCR, or a fragment thereof, composedof at least one TCR α and one TCR β chain sequence, wherein said TCR αchain sequence comprises the CDR1 to CDR3 sequences having the aminoacid sequences of SEQ ID NO: 1 to 3 and 196, and said TCR β chainsequence comprises the CDR1 to CDR3 sequences having the amino acidsequences of SEQ ID NO: 7 to 9; or wherein said TCR α chain sequencecomprises the CDR1 to CDR3 sequences having the amino acid sequences ofSEQ ID NO: 13 to 15 and 197, and said TCR β chain sequence comprises theCDR1 to CDR3 sequences having the amino acid sequences of SEQ ID NO: 19to 21; or wherein said TCR α chain sequence comprises the CDR1 to CDR3sequences having the amino acid sequences of SEQ ID NO: 25 to 27 and198, and said TCR β chain sequence comprises the CDR1 to CDR3 sequenceshaving the amino acid sequences of SEQ ID NO: 31 to 33; or wherein saidTCR α chain sequence comprises the CDR1 to CDR3 sequences having theamino acid sequences of SEQ ID NO: 37 to 39 and 199, and said TCR βchain sequence comprises the CDR1 to CDR3 sequences having the aminoacid sequences of SEQ ID NO: 43 to 45; or wherein said TCR α chainsequence comprises the CDR1 to CDR3 sequences having the amino acidsequences of SEQ ID NO: 49 to 51 and 200, and said TCR β chain sequencecomprises the CDR1 to CDR3 sequences having the amino acid sequences ofSEQ ID NO: 55 to 57; or wherein said TCR α chain sequence comprises theCDR1 to CDR3 sequences having the amino acid sequences of SEQ ID NO: 61to 63 and 201, and said TCR β chain sequence comprises the CDR1 to CDR3sequences having the amino acid sequences of SEQ ID NO: 67 to 69; orwherein said TCR α chain sequence comprises the CDR1 to CDR3 sequenceshaving the amino acid sequences of SEQ ID NO: 73 to 75 and 202, and saidTCR β chain sequence comprises the CDR1 to CDR3 sequences having theamino acid sequences of SEQ ID NO: 79 to 81; or wherein said TCR α chainsequence comprises the CDR1 to CDR3 sequences having the amino acidsequences of SEQ ID NO: 127 to 129 and 204, and said TCR β chainsequence comprises the CDR1 to CDR3 sequences having the amino acidsequences of SEQ ID NO: 133 to 135.

Item 16: The antigen recognizing construct according to any one of items1 to 15, wherein the construct is a TCR, or a fragment thereof,comprising at least one TCR α and one TCR β chain sequence, wherein saidTCR α chain sequence comprises a variable region sequence having theamino acid sequence of SEQ ID No. 4, and wherein said TCR β chainsequence comprises a variable region sequence having the amino acidsequence of SEQ ID No. 10; or wherein said TCR α chain sequencecomprises a variable region sequence having the amino acid sequence ofSEQ ID No. 16, and wherein said TCR β chain sequence comprises avariable region sequence having the amino acid sequence of SEQ ID No.22; or wherein said TCR α chain sequence comprises a variable regionsequence having the amino acid sequence of SEQ ID No. 28, and whereinsaid TCR β chain sequence comprises a variable region sequence havingthe amino acid sequence of SEQ ID No. 34; or wherein said TCR α chainsequence comprises a variable region sequence having the amino acidsequence of SEQ ID No. 40, and wherein said TCR β chain sequencecomprises a variable region sequence having the amino acid sequence ofSEQ ID No. 46; or wherein said TCR α chain sequence comprises a variableregion sequence having the amino acid sequence of SEQ ID No. 52, andwherein said TCR β chain sequence comprises a variable region sequencehaving the amino acid sequence of SEQ ID No. 58; or wherein said TCR αchain sequence comprises a variable region sequence having the aminoacid sequence of SEQ ID No. 64, and wherein said TCR β chain sequencecomprises a variable region sequence having the amino acid sequence ofSEQ ID No. 70; or wherein said TCR α chain sequence comprises a variableregion sequence having the amino acid sequence of SEQ ID No. 76, andwherein said TCR β chain sequence comprises a variable region sequencehaving the amino acid sequence of SEQ ID No. 82; or wherein said TCR αchain sequence comprises a variable region sequence having the aminoacid sequence of SEQ ID No. 130, and wherein said TCR β chain sequencecomprises a variable region sequence having the amino acid sequence ofSEQ ID No. 136.

Item 17: The antigen recognizing construct according to any one of items1 to 16, wherein the construct is a TCR, or a fragment thereof, furthercomprising a TCR constant region having at least 50%, 60%, 70%, 80%,90%, 95%, 98%, 99%, or 100% sequence identity to an amino acid sequenceselected from SEQ ID Nos. 5, 11, 17, 23, 29, 35, 41, 47, 53, 59, 65, 71,77, 83, 131, and 137, preferably wherein the TCR is composed of at leastone TCR α and one TCR β chain sequence, wherein the TCR α chain sequencecomprises a constant region having at least 50%, 60%, 70%, 80%, 90%,95%, 98%, 99%, or 100% sequence identity to an amino acid sequenceselected from SEQ ID Nos. 5, 17, 29, 41, 53, 65, 77, and 131; andwherein the TCR β chain sequence comprises a constant region having atleast 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100% sequence identityto an amino acid sequence selected from SEQ ID Nos. 11, 23, 35, 47, 59,71, 83, and 137.

Item 18: The antigen recognizing construct according to any one of items1 to 17, comprising a first TCR chain having at least 50%, 60%, 70%,80%, 90%, 95%, 98%, 99%, or 100% sequence identity to the amino acidsequence of SEQ ID No. 6, and a second TCR chain having at least 50%,60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100% sequence identity to theamino acid sequence of SEQ ID No. 12.

Item 19: The antigen recognizing construct according to any one of items1 to 17, comprising a first TCR chain having at least 50%, 60%, 70%,80%, 90%, 95%, 98%, 99%, or 100% sequence identity to the amino acidsequence of SEQ ID No. 18, and a second TCR chain having at least 50%,60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100% sequence identity to theamino acid sequence of SEQ ID No. 24.

Item 20a: The antigen recognizing construct according to any one ofitems 1 to 17, comprising a first TCR chain having at least 50%, 60%,70%, 80%, 90%, 95%, 98%, 99%, or 100% sequence identity to the aminoacid sequence of SEQ ID No. 30, and a second TCR chain having at least50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100% sequence identity to theamino acid sequence of SEQ ID No. 36.

Item 20b: The antigen recognizing construct according to any one ofitems 1 to 17, comprising a first TCR chain having at least 50%, 60%,70%, 80%, 90%, 95%, 98%, 99%, or 100% sequence identity to the aminoacid sequence of SEQ ID No. 42, and a second TCR chain having at least50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100% sequence identity to theamino acid sequence of SEQ ID No. 48.

Item 20c: The antigen recognizing construct according to any one ofitems 1 to 17, comprising a first TCR chain having at least 50%, 60%,70%, 80%, 90%, 95%, 98%, 99%, or 100% sequence identity to the aminoacid sequence of SEQ ID No. 54, and a second TCR chain having at least50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100% sequence identity to theamino acid sequence of SEQ ID No. 60.

Item 20d: The antigen recognizing construct according to any one ofitems 1 to 17, comprising a first TCR chain having at least 50%, 60%,70%, 80%, 90%, 95%, 98%, 99%, or 100% sequence identity to the aminoacid sequence of SEQ ID No.66, and a second TCR chain having at least50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100% sequence identity to theamino acid sequence of SEQ ID No. 72.

Item 20e: The antigen recognizing construct according to any one ofitems 1 to 17, comprising a first TCR chain having at least 50%, 60%,70%, 80%, 90%, 95%, 98%, 99%, or 100% sequence identity to the aminoacid sequence of SEQ ID No. 78, and a second TCR chain having at least50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100% sequence identity to theamino acid sequence of SEQ ID No. 84.

Item 20f: The antigen recognizing construct according to any one ofitems 1 to 17, comprising a first TCR chain having at least 50%, 60%,70%, 80%, 90%, 95%, 98%, 99%, or 100% sequence identity to the aminoacid sequence of SEQ ID No. 132, and a second TCR chain having at least50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100% sequence identity to theamino acid sequence of SEQ ID No. 138.

Item 21: A nucleic acid encoding for an antigen recognizing constructaccording to any one of items 1 to 20f.

Item 22: A vector comprising a nucleic acid according to item 21.

Item 23: A host cell comprising an antigen recognizing constructaccording to any one of items 1 to 20, or a nucleic acid according toitem 21, or a vector according to item 22.

Item 24: The host cell according to item 23, wherein the cell is alymphocyte, preferably a T lymphocyte or T lymphocyte progenitor, morepreferably a CD4 or CD8 positive T-cell.

Item 25: A pharmaceutical composition comprising the antigen recognizingconstruct according to any one of items 1 to 20f, or the nucleic acidaccording to item 21, or the vector according to item 22, or the hostcell according to item 23 or 24, and a pharmaceutical acceptablecarrier, stabilizer and/or excipient.

Item 26: The antigen recognizing construct according to any one of items1 to 20f, or a nucleic acid according to item 21, or a vector accordingto item 22, or a host cell according to item 23 or 24, or thepharmaceutical composition according to item 25, for use in medicine.

Item 27: The antigen recognizing construct, or the nucleic acid, or thevector, or the host cell, or the pharmaceutical composition, for useaccording to item 26, for use in the diagnosis, prevention, and/ortreatment of a proliferative disease, wherein the disease comprises amalignant or benign tumor disease.

Item 28: The antigen recognizing construct, or the nucleic acid, or thevector, or the host cell, or the pharmaceutical composition, for useaccording to item 27, wherein the tumor disease is characterized by theexpression of TAA in a tumor cell of the tumor disease.

Item 29: The antigen recognizing construct, or the nucleic acid, or thevector, or the host cell, or the pharmaceutical composition, for useaccording to any one of items 26 to 28, wherein the use in medicine is ause in immune therapy optionally comprising an adoptive cell transfer,wherein the immune therapy comprises adoptive autologous or heterologousT-cell therapy.

Item 30: A method of manufacturing a TAA specific antigen recognizingconstruct expressing cell line, comprising

a., providing a suitable host cell,

b., providing a genetic construct comprising a coding sequence encodingthe antigen recognizing construct according to any one of items 1 to20f,

c., introducing into said suitable host cell said genetic construct,

d., expressing said genetic construct by said suitable host cell.

Item 31: The method according to item 30, further comprising cellsurface presentation of said antigen recognizing construct.

Item 32: The method according to item 30 or 31, wherein the geneticconstruct is an expression construct comprising a promoter sequenceoperably linked to said coding sequence.

Item 33: The method according to any one of items 30 to 32, wherein saidantigen recognizing construct is of mammalian origin, preferably ofhuman origin.

Item 34: The method according to any one of items 30 to 33, wherein saidsuitable host cell is a mammalian cell, optionally selected from a humancell or a human T lymphocyte.

Item 35: The method according to any one of items 30 to 34, wherein saidantigen recognizing construct is a modified TCR, wherein saidmodification comprises addition of a functional domain comprising alabel, or an alternative domain comprising a membrane anchor domain.

Item 36: The method according to item 35, wherein said antigenrecognizing construct is an alpha/beta TCR, gamma/delta TCR, or a singlechain TCR (scTCR).

Item 37: The method according to any one of items 30 to 36, wherein saidgenetic construct is introduced into said suitable host cell byretroviral transfection.

Item 38: The method according to any one of items 30 to 37, furthercomprising the isolation and purification of the antigen recognizingconstruct from the suitable host cell and, optionally, reconstitution ofthe antigen recognizing construct in a T-cell.

The present invention will now be further described in the followingexamples with reference to the accompanying figures and sequences,nevertheless, without being limited thereto. For the purposes of thepresent invention, all references as cited herein are incorporated byreference in their entireties. The Figures and Sequences show:

FIGURES

FIG. 1: IFNγ release from CD8+ T-cells electroporated with alpha andbeta chain RNA of TCR R11P3D3 (Table 1) after co-incubation with T2target cells loaded with PRAME-004 peptide (SEQ ID NO:97) or variousPRAME-004 alanine- or threonine-substitution variants at positions 1-9(X1-X9) of SEQ ID NO:97 (SEQ ID NO:98-115) or control peptide NYESO1-001(SEQ ID NO:126). IFNγ release data were obtained with CD8+ T-cellsderived from two different healthy donors. RNA electroporated CD8+T-cells alone or in co-incubation with unloaded target cells served ascontrols. Several different donors were analyzed with regard toalanine-substitution (Ala_TCRA-0017 and Ala_IFN-041) andthreonine-substitution variants (Thr_TCRA-0036).

FIG. 2: IFNγ release from CD8+ T-cells electroporated with alpha andbeta chain RNA of TCR R16P1C10 (Table 1) after co-incubation with T2target cells loaded with PRAME-004 peptide (SEQ ID NO:97) or variousPRAME-004 alanine- or threonine-substitution variants at positions 1-9(X1-X9) of SEQ ID NO:97 (SEQ ID NO:98-115) or control peptide NYESO1-001(SEQ ID NO:126). IFNγ release data were obtained with CD8+ T-cellsderived from two different healthy donors. RNA electroporated CD8+T-cells alone or in co-incubation with unloaded target cells served ascontrols. Several different donors were analyzed with regard toalanine-substitution (Ala_TCRA-0017 and Ala_IFN-041) andthreonine-substitution variants (Thr_TCRA-0036).

FIG. 3: IFNγ release from CD8+ T-cells electroporated with alpha andbeta chain RNA of TCR R16P1E8 (Table 1) after co-incubation with T2target cells loaded with PRAME-004 peptide (SEQ ID NO:97) or variousPRAME-004 alanine- or threonine-substitution variants at positions 1-9(X1-X9) of SEQ ID NO:97 (SEQ ID NO:98-115) or control peptide NYESO1-001(SEQ ID NO:126). IFNγ release data were obtained with CD8+ T-cellsderived from two different healthy donors. RNA electroporated CD8+T-cells alone or in co-incubation with unloaded target cells served ascontrols. Several different donors were analyzed with regard toalanine-substitution (Ala_TCRA-0017 and Ala_IFN-041) andthreonine-substitution variants (Thr_TCRA-0036).

FIG. 4: IFNγ release from CD8+ T-cells electroporated with alpha andbeta chain RNA of TCR R17P1A9 (Table 1) after co-incubation with T2target cells loaded with PRAME-004 peptide (SEQ ID NO:97) or variousPRAME-004 alanine-substitution variants at positions 1-9 (X1-X9) of SEQID NO:97 (SEQ ID NO:98-106) or control peptide NYESO1-001 (SEQ IDNO:126). IFNγ release data were obtained with CD8+ T-cells derived fromtwo different healthy donors. RNA electroporated CD8+ T-cells alone orin co-incubation with unloaded target cells served as controls.Different donors were analyzed with regard to alanine-substitutionvariants (Ala_IFN-040 and Ala_IFN-041).

FIG. 5: IFNγ release from CD8+ T-cells electroporated with alpha andbeta chain RNA of TCR R17P1D7 (Table 1) after co-incubation with T2target cells loaded with PRAME-004 peptide (SEQ ID NO:97) or variousPRAME-004 alanine- or threonine-substitution variants at positions 1-9(X1-X9) of SEQ ID NO:97 (SEQ ID NO:98-115) or control peptide NYESO1-001(SEQ ID NO:126). IFNγ release data were obtained with CD8+ T-cellsderived from two different healthy donors. RNA electroporated CD8+T-cells alone or in co-incubation with unloaded target cells served ascontrols. Different donors were analyzed with regard toalanine-substitution (Ala_TCRA-0017 and Ala_IFN-041) andthreonine-substitution variants (Thr_TCRA-0036).

FIG. 6: IFNγ release from CD8+ T-cells electroporated with alpha andbeta chain RNA of TCR R17P1G3 (Table 1) after co-incubation with T2target cells loaded with PRAME-004 peptide (SEQ ID NO:97) or variousPRAME-004 alanine- or threonine-substitution variants at positions 1-9(X1-X9) of SEQ ID NO:97 (SEQ ID NO:98-115) or control peptide NYESO1-001(SEQ ID NO:126). IFNγ release data were obtained with CD8+ T-cellsderived from two different healthy donors. RNA electroporated CD8+T-cells alone or in co-incubation with unloaded target cells served ascontrols. Different donors were analyzed with regard toalanine-substitution (Ala_TCRA-0017 and Ala_IFN-041) andthreonine-substitution variants (Thr_TCRA-0036).

FIG. 7: IFNγ release from CD8+ T-cells electroporated with alpha andbeta chain RNA of TCR R17P2B6 (Table 1) after co-incubation with T2target cells loaded with PRAME-004 peptide (SEQ ID NO:97) or variousPRAME-004 alanine- or threonine-substitution variants at positions 1-9(X1-X9) of SEQ ID NO:97 (SEQ ID NO:98-115) or control peptide NYESO1-001(SEQ ID NO:126). IFNγ release data were obtained with CD8+ T-cellsderived from two different healthy donors. RNA electroporated CD8+T-cells alone or in co-incubation with unloaded target cells served ascontrols. Different donors were analyzed with regard toalanine-substitution (Ala_TCRA-0017 and Ala_IFN-041) andthreonine-substitution variants (Thr_TCRA-0036).

FIG. 8: IFNγ release from CD8+ T-cells electroporated with alpha andbeta chain RNA of TCR R11P3D3 (Table 1) after co-incubation with T2target cells loaded with PRAME-004 peptide (SEQ ID NO:97) or similar butunrelated peptide TMED9-001 (SEQ ID NO:116), CAT-001 (SEQ ID NO:117),DDX60L-001 (SEQ ID NO:118), LRRC70-001 (SEQ ID NO:119), PTPLB-001 (SEQID NO:120), HDAC5-001 (SEQ ID NO:121), VPS13B-002 (SEQ ID NO:122),ZNF318-001 (SEQ ID NO:123), CCDC51-001 (SEQ ID NO:124) or IFIT1-001 (SEQID NO:125) or control peptide NYESO1-001 (SEQ ID NO:126). IFNγ releasedata were obtained with CD8+ T-cells derived from two different healthydonors. RNA electroporated CD8+ T-cells alone or in co-incubation withunloaded target cells served as controls. Different donors wereanalyzed, IFN-040 and IFN-041.

FIG. 9: IFNγ release from CD8+ T-cells electroporated with alpha andbeta chain RNA of TCR R16P1C10 (Table 1) after co-incubation with T2target cells loaded with PRAME-004 peptide (SEQ ID NO:97) or similar butunrelated peptide TMED9-001 (SEQ ID NO:116), CAT-001 (SEQ ID NO:117),DDX60L-001 (SEQ ID NO:118), LRRC70-001 (SEQ ID NO:119), PTPLB-001 (SEQID NO:120), HDAC5-001 (SEQ ID NO:121), VPS13B-002 (SEQ ID NO:122),ZNF318-001 (SEQ ID NO:123), CCDC51-001 (SEQ ID NO:124) or IFIT1-001 (SEQID NO:125) or control peptide NYESO1-001 (SEQ ID NO:126). IFNγ releasedata were obtained with CD8+ T-cells derived from two different healthydonors. RNA electroporated CD8+ T-cells alone or in co-incubation withunloaded target cells served as controls. Different donors wereanalyzed, IFN-046 and IFN-041.

FIG. 10: IFNγ release from CD8+ T-cells electroporated with alpha andbeta chain RNA of TCR R16P1E8 (Table 1) after co-incubation with T2target cells loaded with PRAME-004 peptide (SEQ ID NO:97) or similar butunrelated peptide TMED9-001 (SEQ ID NO:116), CAT-001 (SEQ ID NO:117),DDX60L-001 (SEQ ID NO:118), LRRC70-001 (SEQ ID NO:119), PTPLB-001 (SEQID NO:120), HDAC5-001 (SEQ ID NO:121), VPS13B-002 (SEQ ID NO:122),ZNF318-001 (SEQ ID NO:123), CCDC51-001 (SEQ ID NO:124) or IFIT1-001 (SEQID NO:125) or control peptide NYESO1-001 (SEQ ID NO:126). IFNγ releasedata were obtained with CD8+ T-cells derived from two different healthydonors. RNA electroporated CD8+ T-cells alone or in co-incubation withunloaded target cells served as controls. Different donors wereanalyzed, IFN-040 and IFN-041.

FIG. 11: IFNγ release from CD8+ T-cells electroporated with alpha andbeta chain RNA of TCR R17P1A9 (Table 1) after co-incubation with T2target cells loaded with PRAME-004 peptide (SEQ ID NO:97) or similar butunrelated peptide TMED9-001 (SEQ ID NO:116), CAT-001 (SEQ ID NO:117),DDX60L-001 (SEQ ID NO:118), LRRC70-001 (SEQ ID NO:119), PTPLB-001 (SEQID NO:120), HDAC5-001 (SEQ ID NO:121), VPS13B-002 (SEQ ID NO:122),ZNF318-001 (SEQ ID NO:123), CCDC51-001 (SEQ ID NO:124) or IFIT1-001 (SEQID NO:125) or control peptide NYESO1-001 (SEQ ID NO:126). IFNγ releasedata were obtained with CD8+ T-cells derived from two different healthydonors. RNA electroporated CD8+ T-cells alone or in co-incubation withunloaded target cells served as controls. Different donors wereanalyzed, IFN-040 and IFN-041.

FIG. 12: IFNγ release from CD8+ T-cells electroporated with alpha andbeta chain RNA of TCR R17P1D7 (Table 1) after co-incubation with T2target cells loaded with PRAME-004 peptide (SEQ ID NO:97) or similar butunrelated peptide TMED9-001 (SEQ ID NO:116), CAT-001 (SEQ ID NO:117),DDX60L-001 (SEQ ID NO:118), LRRC70-001 (SEQ ID NO:119), PTPLB-001 (SEQID NO:120), HDAC5-001 (SEQ ID NO:121), VPS13B-002 (SEQ ID NO:122),ZNF318-001 (SEQ ID NO:123), CCDC51-001 (SEQ ID NO:124) or IFIT1-001 (SEQID NO:125) or control peptide NYESO1-001 (SEQ ID NO:126). IFNγ releasedata were obtained with CD8+ T-cells derived from two different healthydonors. RNA electroporated CD8+ T-cells alone or in co-incubation withunloaded target cells served as controls. Different donors wereanalyzed, IFN-040 and IFN-041.

FIG. 13: IFNγ release from CD8+ T-cells electroporated with alpha andbeta chain RNA of TCR R17P1G3 (Table 1) after co-incubation with T2target cells loaded with PRAME-004 peptide (SEQ ID NO:97) or similar butunrelated peptide TMED9-001 (SEQ ID NO:116), CAT-001 (SEQ ID NO:117),DDX60L-001 (SEQ ID NO:118), LRRC70-001 (SEQ ID NO:119), PTPLB-001 (SEQID NO:120), HDAC5-001 (SEQ ID NO:121), VPS13B-002 (SEQ ID NO:122),ZNF318-001 (SEQ ID NO:123), CCDC51-001 (SEQ ID NO:124) or IFIT1-001 (SEQID NO:125) or control peptide NYESO1-001 (SEQ ID NO:126). IFNγ releasedata were obtained with CD8+ T-cells derived from two different healthydonors. RNA electroporated CD8+ T-cells alone or in co-incubation withunloaded target cells served as controls. Different donors wereanalyzed, IFN-046 and IFN-041.

FIG. 14: IFNγ release from CD8+ T-cells electroporated with alpha andbeta chain RNA of TCR R17P2B6 (Table 1) after co-incubation with T2target cells loaded with PRAME-004 peptide (SEQ ID NO:97) or similar butunrelated peptide TMED9-001 (SEQ ID NO:116), CAT-001 (SEQ ID NO:117),DDX60L-001 (SEQ ID NO:118), LRRC70-001 (SEQ ID NO:119), PTPLB-001 (SEQID NO:120), HDAC5-001 (SEQ ID NO:121), VPS13B-002 (SEQ ID NO:122),ZNF318-001 (SEQ ID NO:123), CCDC51-001 (SEQ ID NO:124) or IFIT1-001 (SEQID NO:125) or control peptide NYESO1-001 (SEQ ID NO:126). IFNγ releasedata were obtained with CD8+ T-cells derived from two different healthydonors. RNA electroporated CD8+ T-cells alone or in co-incubation withunloaded target cells served as controls. Different donors wereanalyzed, IFN-040 and IFN-041.

FIG. 15: IFNγ release from CD8+ T-cells electroporated with alpha andbeta chain RNA of TCR R11P3D3 (Table 1) after co-incubation with T2target cells loaded with PRAME-004 peptide (SEQ ID NO:97) in variouspeptide loading concentrations from 10 μM to 10 pM. IFNγ release datawere obtained with CD8+ T-cells derived from two different healthydonors. Different donors were analyzed, TCRA-0003 and TCRA-0017.

FIG. 16: IFNγ release from CD8+ T-cells electroporated with alpha andbeta chain RNA of TCR R16P1C10 (Table 1) after co-incubation with T2target cells loaded with PRAME-004 peptide (SEQ ID NO:97) in variouspeptide loading concentrations from 10 μM to 10 pM. IFNγ release datawere obtained with CD8+ T-cells derived from two different healthydonors. Different donors were analyzed, TCRA-0003 and TCRA-0017.

FIG. 17: IFNγ release from CD8+ T-cells electroporated with alpha andbeta chain RNA of TCR R16P1E8 (Table 1) after co-incubation with T2target cells loaded with PRAME-004 peptide (SEQ ID NO:97) in variouspeptide loading concentrations from 10 μM to 10 pM. IFNγ release datawere obtained with CD8+ T-cells derived from two different healthydonors. Different donors were analyzed, TCRA-0003 and TCRA-0017.

FIG. 18: IFNγ release from CD8+ T-cells electroporated with alpha andbeta chain RNA of TCR R17P1D7 (Table 1) after co-incubation with T2target cells loaded with PRAME-004 peptide (SEQ ID NO:97) in variouspeptide loading concentrations from 10 μM to 10 pM. IFNγ release datawere obtained with CD8+ T-cells derived from two different healthydonors. Different donors were analyzed, TCRA-0003 and TCRA-0017.

FIG. 19: IFNγ release from CD8+ T-cells electroporated with alpha andbeta chain RNA of TCR R17P1G3 (Table 1) after co-incubation with T2target cells loaded with PRAME-004 peptide (SEQ ID NO:97) in variouspeptide loading concentrations from 10 μM to 10 pM. IFNγ release datawere obtained with CD8+ T-cells derived from two different healthydonors. Different donors were analyzed, TCRA-0003 and TCRA-0017.

FIG. 20: IFNγ release from CD8+ T-cells electroporated with alpha andbeta chain RNA of TCR R17P2B6 (Table 1) after co-incubation with T2target cells loaded with PRAME-004 peptide (SEQ ID NO:97) in variouspeptide loading concentrations from 10 μM to 10 pM. IFNγ release datawere obtained with CD8+ T-cells derived from two different healthydonors. Different donors were analyzed, TCRA-0003 and TCRA-0017.

FIG. 21: HLA-A*02/PRAME-004 tetramer or HLA-A*02/NYESO1-001 tetramerstaining, respectively, of CD8+ T-cells electroporated with alpha andbeta chain RNA of TCR R16P1C10 (Table 1). CD8+ T-cells electroporatedwith RNA of 1G4 TCR (SEQ ID: 85-96) that specifically binds to theHLA-A*02/NYESO1-001 complex and mock electroporated CD8+ T-cells servedas controls.

FIG. 22: IFNγ release from CD8+ T-cells lentivirally transduced with TCRR11P3D3 (Table 1) (D103805 and D191451) or non-transduced cells (D103805NT and D191451 NT) after co-incubation with T2 target cells loaded with100 nM PRAME-004 peptide (SEQ ID NO:97) or similar (identical toPRAME-004 in positions 3, 5, 6 and 7) but unrelated peptides ACPL-001(SEQ ID NO:139), HSPB3-001 (SEQ ID NO:140), UNC7-001 (SEQ ID NO:141),SCYL2-001 (SEQ ID NO:142), RPS2P8-001 (SEQ ID NO:143), PCNXL3-003 (SEQID NO:144), AQP6-001 (SEQ ID NO:145), PCNX-001 (SEQ ID NO:146), AQP6-002(SEQ ID NO:147) TRGV10-001 (SEQ ID NO:148), NECAP1-001 (SEQ ID NO:149)or FBXW2-001 (SEQ ID NO:150) or control peptide NYESO1-001 (SEQ IDNO:126). IFNγ release data were obtained with CD8+ T-cells derived fromtwo different healthy donors, D103805 and D191451.

FIG. 23: IFNγ release from CD8+ T-cells lentivirally transduced with TCRR11P3D3 (Table 1) after co-incubation with T2 target cells loaded with100 nM PRAME-004 peptide (SEQ ID NO:97) or similar (identical toPRAME-004 in positions 3, 5, 6 and 7) but unrelated peptides (SEQ IDNO:151-195) or control peptide NYESO1-001 (SEQ ID NO:126). IFNγ releasedata were obtained with CD8+ T-cells derived from two different healthydonors, TCRA-0087 and TCRA-0088.

FIG. 24: IFNγ release from CD8+ T-cells lentivirally transduced with TCRR11P3D3 (Table 1) (D103805 and D191451) or non-transduced cells (D103805NT and D191451 NT) after co-incubation with different primary cells(HCASMC (Coronary artery smooth muscle cells), HTSMC (Tracheal smoothmuscle cells), HRCEpC (Renal cortical epithelial cells), HCM(Cardiomyocytes), HCMEC (Cardiac microvascular endothelial cells),HSAEpC (Small airway epithelial cells), HCF (Cardiac fibroblasts)) andiPSC-derived cell types (HN (Neurons), iHCM (Cardiomyocytes), HH(Hepatocytes), HA (astrocytes)). Tumor cell lines UACC-257 (PRAME-004high), Hs695T (PRAME-004 medium), U266B1 (PRAME-004 very low) and MCF-7(no PRAME-004) present different amounts of PRAME-004 per cells. T-cellsalone served as controls. IFNγ release data were obtained with CD8+T-cells derived from two different healthy donors, D103805 and D191451.

FIG. 25: IFNγ release from CD8+ T-cells lentivirally transduced with TCRR11P3D3 (Table 1) after co-incubation with different primary cells (NHEK(Epidermal keratinocytes), HBEpC (Bronchial epithelial cells), HDMEC(Dermal microvascular endothelial cells), HCAEC (Coronary arteryendothelial cells), HAoEC (Aortic endothelial cells), HPASMC (Pulmonaryartery smooth muscle cells), HAoSMC (Aortic smooth muscle cells), HPF(Pulmonary fibroblasts), SkMC (Skeletal muscle cells), HOB(osteoblasts), HCH (Chondrocytes), HWP (White preadipocytes), hMSC-BM(Mesenchymal stem cells), NHDF (Dermal fibroblasts). Tumor cell linesUACC-257 (PRAME-004 high), Hs695T (PRAME-004 medium), U266B1 (PRAME-004very low) and MCF-7 (no PRAME-004) present different copies of PRAME-004per cells. T-cells alone served as controls. IFNγ release data wereobtained with CD8+ T-cells derived from two different healthy donors,TCRA-0084 and TCRA-0085.

FIG. 26: IFNγ release from CD8+ T-cells lentivirally transduced withenhanced TCR R11P3D3_KE (Table 1) (D103805 and D191451) ornon-transduced cells (D103805 NT and D191451 NT) after co-incubationwith T2 target cells loaded with 100 nM PRAME-004 peptide (SEQ ID NO:97)or similar (identical to PRAME-004 in positions 3, 5, 6 and 7) butunrelated peptide ACPL-001 (SEQ ID NO:139), HSPB3-001 (SEQ ID NO:140),UNC7-001 (SEQ ID NO:141), SCYL2-001 (SEQ ID NO:142), RPS2P8-001 (SEQ IDNO:143), PCNXL3-003 (SEQ ID NO:144), AQP6-001 (SEQ ID NO:145), PCNX-001(SEQ ID NO:146), AQP6-002 (SEQ ID NO:147), TRGV10-001 (SEQ ID NO:148),NECAP1-001 (SEQ ID NO:149) or FBXW2-001 (SEQ ID NO:150) or controlpeptide NYESO1-001 (SEQ ID NO:126). IFNγ release data were obtained withCD8+ T-cells derived from two different healthy donors, D103805 andD191451.

FIG. 27: IFNγ release from CD8+ T-cells lentivirally transduced withenhanced TCR R11P3D3_KE (Table 1) after co-incubation with T2 targetcells loaded with 100 nM PRAME-004 peptide (SEQ ID NO:97) or similar(identical to PRAME-004 in positions 3, 5, 6 and 7) but unrelatedpeptides (SEQ ID NO:151-195) or control peptide NYESO1-001 (SEQ IDNO:126). IFNγ release data were obtained with CD8+ T-cells derived fromtwo different healthy donors, TCRA-0087 and TCRA-0088.

FIG. 28: IFNγ release from CD8+ T-cells lentivirally transduced withenhanced TCR R11P3D3_KE (Table 1) (D103805 and D191451) ornon-transduced cells (D103805 NT and D191451 NT) after co-incubationwith different primary cells (HCASMC (Coronary artery smooth musclecells), HTSMC (Tracheal smooth muscle cells), HRCEpC (Renal corticalepithelial cells), HCM (Cardiomyocytes), HCMEC (Cardiac microvascularendothelial cells), HSAEpC (Small airway epithelial cells), HCF (Cardiacfibroblasts)) and iPSC-derived cell types (HN (Neurons), iHCM(Cardiomyocytes), HH (Hepatocytes), HA (astrocytes)). Tumor cell linesUACC-257 (PRAME-004 high), Hs695T (PRAME-004 medium), U266B1 (PRAME-004very low) and MCF-7 (no PRAME-004) present different amounts ofPRAME-004 per cells. T-cells alone served as controls. IFNγ release datawere obtained with CD8+ T-cells derived from two different healthydonors, D103805 and D191451.

FIG. 29: IFNγ release from CD8+ T-cells lentivirally transduced withenhanced TCR R11P3D3_KE (Table 1) after co-incubation with differentprimary cells (NHEK (Epidermal keratinocytes), HBEpC (Bronchialepithelial cells), HDMEC (Dermal microvascular endothelial cells), HCAEC(Coronary artery endothelial cells), HAoEC (Aortic endothelial cells),HPASMC (Pulmonary artery smooth muscle cells), HAoSMC (Aortic smoothmuscle cells), HPF (Pulmonary fibroblasts), SkMC (Skeletal musclecells), HOB (osteoblasts), HCH (Chondrocytes), HWP (Whitepreadipocytes), hMSC-BM (Mesenchymal stem cells), NHDF (Dermalfibroblasts). Tumor cell lines UACC-257 (PRAME-004 high), Hs695T(PRAME-004 medium), U266B1 (PRAME-004 very low) and MCF-7 (no PRAME-004)present different copies of PRAME-004 per cells. T-cells alone served ascontrols. IFNγ release data were obtained with CD8+ T-cells derived fromtwo different healthy donors, TCRA-0084 and TCRA-0085.

FIG. 30: IFNγ release from CD8+ T-cells lentivirally transduced with TCRR11P3D3 or enhanced TCR R11P3D3_KE (Table 1) or non-transduced cellsafter co-incubation with tumor cell lines UACC-257 (PRAME-004 high),Hs695T (PRAME-004 medium), U266B1 (PRAME-004 very low) and MCF-7 (noPRAME-004) present different amounts of PRAME-004 per cells. T-cellsalone served as controls. IFNγ release of both TCRs correlates withPRAME-004 presentation and R11P3D3_KE induces higher responses comparedto R11P3D3.

FIG. 31: IFNγ release from CD8+ T-cells lentivirally transduced withenhanced TCR R11P3D3_KE (Table 1) cells after co-incubation with T2target cells loaded with various PRAME-004 alanine-substitution variantsat positions 1-9 (A1-A9) of SEQ ID NO:97 (SEQ ID NO:98-106). IFNγrelease data were obtained with CD8+ T-cells derived from threedifferent healthy donors.

FIG. 32: Potency assay evaluating cytolytic activity of lentivirallytransduced T cells expressing TCR R11P3D3 or enhanced TCR R11P3D3_KEagainst PRAME-004+ tumor cells. Cytotoxic response of R11P3D3 andR11P3D3_KE transduced and non-transduced (NT) T cells measured againstA-375 (PRAME-004 low) or U2OS (PRAME-004 medium) tumor cells. The assayswere performed in a 72-hour fluorescence microscopy-based cytotoxicityassay. Results are shown as fold tumor growth over time.

TABLE 1 TCR sequences of the invention SEQ ID NO: TCR Chain RegionSequence 1 R11P3D3 alpha CDR1 SSNFYA 2 R11P3D3 alpha CDR2 MTL 3 R11P3D3alpha CDR3 CALYNNNDMRF 4 R11P3D3 alpha variableMEKNPLAAPLLILWFHLDCVSSILNVEQSPQSLHVQEGDSTNFTCSFPSSNFYALHWYRWETA domainKSPEALFVMTLNGDEKKKGRISATLNTKEGYSYLYIKGSQPEDSATYLCALYNNNDMRFGAGT RLTVKP 5R11P3D3 alpha constantNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAV domainAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS 6 R11P3D3 alpha full-MEKNPLAAPLLILWFHLDCVSSILNVEQSPQSLHVQEGDSTNFTCSFPSSNFYALHWYRWETA lengthKSPEALFVMTLNGDEKKKGRISATLNTKEGYSYLYIKGSQPEDSATYLCALYNNNDMRFGAGTRLTVKPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS 7 R11P3D3 beta CDR1 SGHNS 8 R11P3D3 beta CDR2 FNNNVP9 R11P3D3 beta CDR3 CASSPGSTDTQYF 10 R11P3D3 beta variableMDSWTFCCVSLCILVAKHTDAGVIQSPRHEVTEMGQEVTLRCKPISGHNSLFWYRQTMMRGL domainELLIYFNNNVPIDDSGMPEDRFSAKMPNASFSTLKIQPSEPRDSAVYFCASSPGSTDTQYFGPG TRLTVL11 R11P3D3 beta constantEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPL domainKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG 12 R11P3D3 betafull- MDSWTFCCVSLCILVAKHTDAGVIQSPRHEVTEMGQEVTLRCKPISGHNSLFVVYRQTMMRGLlength ELLIYFNNNVPIDDSGMPEDRFSAKMPNASFSTLKIQPSEPRDSAVYFCASSPGSTDTQYFGPGTRLTVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG 13R16P1C10 alpha CDR1 DRGSQS 14 R16P1C10 alpha CDR2 IY 15 R16P1C10 alphaCDR3 CAAVISNFGNEKLTF 16 R16P1C10 alpha variableMKSLRVLLVILWLQLSWVWSQQKEVEQNSGPLSVPEGAIASLNCTYSDRGSQSFFWYRQYSG domainKSPELIMFIYSNGDKEDGRFTAQLNKASQYVSLLIRDSQPSDSATYLCAAVISNFGNEKLTFGTGTRLTIIP 17 R16P1C10 alpha constantNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAV domainAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS 18 R16P1C10 alpha full-MKSLRVLLVILWLQLSWVWSQQKEVEQNSGPLSVPEGAIASLNCTYSDRGSQSFFWYRQYSG lengthKSPELIMFIYSNGDKEDGRFTAQLNKASQYVSLLIRDSQPSDSATYLCAAVISNFGNEKLTFGTGTRLTIIPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS 19 R16P1C10 beta CDR1 SGHRS 20 R16P1C10 beta CDR2YFSETQ 21 R16P1C10 beta CDR3 CASSPWDSPNEQYF 22 R16P1C10 beta variableMGSRLLCWVLLCLLGAGPVKAGVTQTPRYLIKTRGQQVTLSCSPISGHRSVSWYQQTPGQGL domainQFLFEYFSETQRNKGNFPGRFSGRQFSNSRSEMNVSTLELGDSALYLCASSPWDSPNEQYFG PGTRLTVT23 R16P1C10 beta constantEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPL domainKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG 24 R16P1C10 betafull- MGSRLLCWVLLCLLGAGPVKAGVTQTPRYLIKTRGQQVTLSCSPISGHRSVSWYQQTPGQGLlength QFLFEYFSETQRNKGNFPGRFSGRQFSNSRSEMNVSTLELGDSALYLCASSPWDSPNEQYFGPGTRLTVTEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG 25R16P1E8 alpha CDR1 NSAFQY 26 R16P1E8 alpha CDR2 TY 27 R16P1E8 alpha CDR3CAMSEAAGNKLTF 28 R16P1E8 alpha variableMMKSLRVLLVILWLQLSWVWSQQKEVEQDPGPLSVPEGAIVSLNCTYSNSAFQYFMWYRQYS domainRKGPELLMYTYSSGNKEDGRFTAQVDKSSKYISLFIRDSQPSDSATYLCAMSEAAGNKLTFGG GTRVLVKP29 R16P1E8 alpha constantNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAV domainAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS 30 R16P1E8 alpha full-MMKSLRVLLVILWLQLSWVWSQQKEVEQDPGPLSVPEGAIVSLNCTYSNSAFQYFMWYRQYS lengthRKGPELLMYTYSSGNKEDGRFTAQVDKSSKYISLFIRDSQPSDSATYLCAMSEAAGNKLTFGGGTRVLVKPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS 31 R16P1E8 beta CDR1 SGHAT 32 R16P1E8 beta CDR2FQNNGV 33 R16P1E8 beta CDR3 CASSYTNQGEAFF 34 R16P1E8 beta variableMGTRLLCWAALCLLGAELTEAGVAQSPRYKIIEKRQSVAFWCNPISGHATLYWYQQILGQGPK domainLLIQFQNNGVVDDSQLPKDRFSAERLKGVDSTLKIQPAKLEDSAVYLCASSYTNQGEAFFGQG TRLTVV35 R16P1E8 beta constantEDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPL domainKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF 36 R16P1E8 betafull- MGTRLLCWAALCLLGAELTEAGVAQSPRYKIIEKRQSVAFWCNPISGHATLYWYQQILGQGPKlength LLIQFQNNGVVDDSQLPKDRFSAERLKGVDSTLKIQPAKLEDSAVYLCASSYTNQGEAFFGQGTRLTVVEDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF 37 R17P1A9alpha CDR1 DRGSQS 38 R17P1A9 alpha CDR2 IY 39 R17P1A9 alpha CDR3CAVLNQAGTALIF 40 R17P1A9 alpha variableMKSLRVLLVILWLQLSWVWSQQKEVEQNSGPLSVPEGAIASLNCTYSDRGSQSFFWYRQYSG domainKSPELIMSIYSNGDKEDGRFTAQLNKASQYVSLLIRDSQPSDSATYLCAVLNQAGTALIFGKGTT LSVSS41 R17P1A9 alpha constantNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAV domainAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS 42 R17P1A9 alpha full-MKSLRVLLVILWLQLSWVWSQQKEVEQNSGPLSVPEGAIASLNCTYSDRGSQSFFWYRQYSG lengthKSPELIMSIYSNGDKEDGRFTAQLNKASQYVSLLIRDSQPSDSATYLCAVLNQAGTALIFGKGTTLSVSSNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS 43 R17P1A9 beta CDR1 SGDLS 44 R17P1A9 beta CDR2YYNGEE 45 R17P1A9 beta CDR3 CASSAETGPWLGNEQFF 46 R17P1A9 beta variableMGFRLLCCVAFCLLGAGPVDSGVTQTPKHLITATGQRVTLRCSPRSGDLSVYWYQQSLDQGL domainQFLIQYYNGEERAKGNILERFSAQQFPDLHSELNLSSLELGDSALYFCASSAETGPWLGNEQFFGPGTRLTVL 47 R17P1A9 beta constantEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPL domainKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG 48 R17P1A9 betafull- MGFRLLCCVAFCLLGAGPVDSGVTQTPKHLITATGQRVTLRCSPRSGDLSVYWYQQSLDQGLlength QFLIQYYNGEERAKGNILERFSAQQFPDLHSELNLSSLELGDSALYFCASSAETGPWLGNEQFFGPGTRLTVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG 49R17P1D7 alpha CDR1 TSESDYY 50 R17P1D7 alpha CDR2 QEAY 51 R17P1D7 alphaCDR3 CAYRWAQGGSEKLVF 52 R17P1D7 alpha variableMACPGFLWALVISTCLEFSMAQTVTQSQPEMSVQEAETVTLSCTYDTSESDYYLFWYKQPPS domainRQMILVIRQEAYKQQNATENRFSVNFQKAAKSFSLKISDSQLGDAAMYFCAYRWAQGGSEKLVFGKGTKLTVNP 53 R17P1D7 alpha constantYIQKPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAV domainAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS 54 R17P1D7 alpha full-MACPGFLWALVISTCLEFSMAQTVTQSQPEMSVQEAETVTLSCTYDTSESDYYLFWYKQPPS lengthRQMILVIRQEAYKQQNATENRFSVNFQKAAKSFSLKISDSQLGDAAMYFCAYRWAQGGSEKLVFGKGTKLTVNPYIQKPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS 55 R17P1D7 beta CDR1 MGHDK 56 R17P1D7 betaCDR2 SYGVNS 57 R17P1D7 beta CDR3 CATELWSSGGTGELFF 58 R17P1D7 betavariable MTIRLLCYMGFYFLGAGLMEADIYQTPRYLVIGTGKKITLECSQTMGHDKMYWYQQDPGMELHdomain LIHYSYGVNSTEKGDLSSESTVSRIRTEHFPLTLESARPSHTSQYLCATELWSSGGTGELFFGEGSRLTVL 59 R17P1D7 beta constantEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPL domainKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG 60 R17P1D7 betafull- MTIRLLCYMGFYFLGAGLMEADIYQTPRYLVIGTGKKITLECSQTMGHDKMYWYQQDPGMELHlength LIHYSYGVNSTEKGDLSSESTVSRIRTEHFPLTLESARPSHTSQYLCATELWSSGGTGELFFGEGSRLTVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG 61R17P1G3 alpha CDR1 DRGSQS 62 R17P1G3 alpha CDR2 IY 63 R17P1G3 alpha CDR3CAVGPSGTYKYIF 64 R17P1G3 alpha variableMKSLRVLLVILWLQLSWVWSQQKEVEQNSGPLSVPEGAIASLNCTYSDRGSQSFFWYRQYSG domainKSPELIMSIYSNGDKEDGRFTAQLNKASQYVSLLIRDSQPSDSATYLCAVGPSGTYKYIFGTGT RLKVLA65 R17P1G3 alpha constantNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAV domainAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS 66 R17P1G3 alpha full-MKSLRVLLVILWLQLSWVWSQQKEVEQNSGPLSVPEGAIASLNCTYSDRGSQSFFWYRQYSG lengthKSPELIMSIYSNGDKEDGRFTAQLNKASQYVSLLIRDSQPSDSATYLCAVGPSGTYKYIFGTGTRLKVLANIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS 67 R17P1G3 beta CDR1 MNHEY 68 R17P1G3 beta CDR2SMNVEV 69 R17P1G3 beta CDR3 CASSPGGSGNEQFF 70 R17P1G3 beta variableMGPQLLGYVVLCLLGAGPLEAQVTQNPRYLITVTGKKLTVTCSQNMNHEYMSWYRQDPGLGL domainRQIYYSMNVEVTDKGDVPEGYKVSRKEKRNFPLILESPSPNQTSLYFCASSPGGSGNEQFFGP GTRLTVL71 R17P1G3 beta constantEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPL domainKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG 72 R17P1G3 betafull- MGPQLLGYVVLCLLGAGPLEAQVTQNPRYLITVTGKKLTVTCSQNMNHEYMSWYRQDPGLGLlength RQIYYSMNVEVTDKGDVPEGYKVSRKEKRNFPLILESPSPNQTSLYFCASSPGGSGNEQFFGPGTRLTVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG 73R17P2B6 alpha CDR1 DRGSQS 74 R17P2B6 alpha CDR2 IY 75 R17P2B6 alpha CDR3CAVVSGGGADGLTF 76 R17P2B6 alpha variableMKSLRVLLVILWLQLSWVWSQQKEVEQNSGPLSVPEGAIASLNCTYSDRGSQSFFWYRQYSG domainKSPELIMFIYSNGDKEDGRFTAQLNKASQYVSLLIRDSQPSDSATYLCAVVSGGGADGLTFGKG THLIIQP77 R17P2B6 alpha constantYIQKPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAV domainAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS 78 R17P2B6 alpha full-MKSLRVLLVILWLQLSWVWSQQKEVEQNSGPLSVPEGAIASLNCTYSDRGSQSFFWYRQYSG lengthKSPELIMFIYSNGDKEDGRFTAQLNKASQYVSLLIRDSQPSDSATYLCAVVSGGGADGLTFGKGTHLIIQPYIQKPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS 79 R17P2B6 beta CDR1 PRHDT 80 R17P2B6 beta CDR2FYEKMQ 81 R17P2B6 beta CDR3 CASSLGRGGQPQHF 82 R17P2B6 beta variableMLSPDLPDSAWNTRLLCHVMLCLLGAVSVAAGVIQSPRHLIKEKRETATLKCYPIPRHDTVYWY domainQQGPGQDPQFLISFYEKMQSDKGSIPDRFSAQQFSDYHSELNMSSLELGDSALYFCASSLGRGGQPQHFGDGTRLSIL 83 R17P2B6 beta constantEDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPL domainKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF 84 R17P2B6 betafull- MLSPDLPDSAWNTRLLCHVMLCLLGAVSVAAGVIQSPRHLIKEKRETATLKCYPIPRHDTVYWYlength QQGPGQDPQFLISFYEKMQSDKGSIPDRFSAQQFSDYHSELNMSSLELGDSALYFCASSLGRGGQPQHFGDGTRLSILEDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMV KRKDF85 1G4 alpha CDR1 DSAIYN 86 1G4 alpha CDR2 IQS 87 1G4 alpha CDR3CAVRPTSGGSYIPTF 88 1G4 alpha variableMETLLGLLILWLQLQWVSSKQEVTQIPAALSVPEGENLVLNCSFTDSAIYNLQWFRQDPGKGLT domainSLLLIQSSQREQTSGRLNASLDKSSGRSTLYIAASQPGDSATYLCAVRPTSGGSYIPTFGRGTS LIVHP89 1G4 alpha constantYIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAV domainAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS 90 1G4 alpha full-METLLGLLILWLQLQWVSSKQEVTQIPAALSVPEGENLVLNCSFTDSAIYNLQWFRQDPGKGLT lengthSLLLIQSSQREQTSGRLNASLDKSSGRSTLYIAASQPGDSATYLCAVRPTSGGSYIPTFGRGTSLIVHPYIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS 91 1G4 beta CDR1 MNHEY 92 1G4 beta CDR2 SVGAGI 93 1G4beta CDR3 CASSYVGNTGELFF 94 1G4 beta variableMSIGLLCCAALSLLWAGPVNAGVTQTPKFQVLKTGQSMTLQCAQDMNHEYMSWYRQDPGMG domainLRLIHYSVGAGITDQGEVPNGYNVSRSTTEDFPLRLLSAAPSQTSVYFCASSYVGNTGELFFGE GSRLTVL95 1G4 beta constantEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPL domainKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG 96 1G4 beta full-MSIGLLCCAALSLLWAGPVNAGVTQTPKFQVLKTGQSMTLQCAQDMNHEYMSWYRQDPGMG lengthLRLIHYSVGAGITDQGEVPNGYNVSRSTTEDFPLRLLSAAPSQTSVYFCASSYVGNTGELFFGEGSRLTVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG 127R11P3D3_ alpha CDR1 SSNFYA KE 128 R11P3D3_ alpha CDR2 MTL KE 129R11P3D3_ alpha CDR3 CALYNNNDMRF KE 130 R11P3D3_ alpha variableMEKNPLAAPLLILWFHLDCVSSILNVEQSPQSLHVQEGDSTNFTCSFPSSNFYALHWYRKETAK KEdomain SPEALFVMTLNGDEKKKGRISATLNTKEGYSYLYIKGSQPEDSATYLCALYNNNDMRFGAGTRLTVKP 131 R11P3D3_ alpha constantNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAV KEdomain AWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS 132 R11P3D3_ alpha full-MEKNPLAAPLLILWFHLDCVSSILNVEQSPQSLHVQEGDSTNFTCSFPSSNFYALHWYRKETAK KElength SPEALFVMTLNGDEKKKGRISATLNTKEGYSYLYIKGSQPEDSATYLCALYNNNDMRFGAGTRLTVKPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS 133 R11P3D3_ beta CDR1 SGHNS KE 134 R11P3D3_ beta CDR2FNNNVP KE 135 R11P3D3_ beta CDR3 CASSPGSTDTQYF KE 136 R11P3D3_ betavariable MDSWTFCCVSLCILVAKHTDAGVIQSPRHEVTEMGQEVTLRCKPISGHNSLFWYRETMMRGLKE domainELLIYFNNNVPIDDSGMPEDRFSAKMPNASFSTLKIQPSEPRDSAVYFCASSPGSTDTQYFGPG TRLTVL137 R11P3D3_ beta constantEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPL KEdomain KEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEVVTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG 138 R11P3D3_ betafull- MDSWTFCCVSLCILVAKHTDAGVIQSPRHEVTEMGQEVTLRCKPISGHNSLFWYRETMMRGL KEdomain ELLIYFNNNVPIDDSGMPEDRFSAKMPNASFSTLKIQPSEPRDSAVYFCASSPGSTDTQYFGPGTRLTVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVT 196R11P3D3 alpha CDR2bi MTLNGDE 197 R16P1C10 alpha CDR2bi IYSNGD 198R16P1E8 alpha CDR2bi TYSSGN 199 R17P1A9 alpha CDR2bi IYSNGD 200 R17P1D7alpha CDR2bi QEAYKQQ 201 R17P1G3 alpha CDR2bi IYSNGD 202 R17P2B6 alphaCDR2bi IYSNGD 203 1G4 alpha CDR2bi IQSSQRE 204 R11P3D3__ alpha CDR2biMTLNGDE

TABLE 2 Peptide sequences of the invention Peptide Code SequenceSEQ ID NO: PRAME-004 SLLQHLIGL 97 PRAME-004_A1 ALLQHLIGL 98 PRAME-004_A2SALQHLIGL 99 PRAME-004_A3 SLAQHLIGL 100 PRAME-004_A4 SLLAHLIGL 101PRAME-004_A5 SLLQALIGL 102 PRAME-004_A6 SLLQHAIGL 103 PRAME-004_A7SLLQHLAGL 104 PRAME-004_A8 SLLQHLIAL 105 PRAME-004_A9 SLLQHLIGA 106PRAME-004_T1 TLLQHLIGL 107 PRAME-004_T2 STLQHLIGL 108 PRAME-004_T3SLTQHLIGL 109 PRAME-004_T4 SLLTHLIGL 110 PRAME-004_T5 SLLQTLIGL 111PRAME-004_T6 SLLQHTIGL 112 PRAME-004_T7 SLLQHLTGL 113 PRAME-004_T8SLLQHLITL 114 PRAME-004_T9 SLLQHLIGT 115 TMED9-001 SILQTLILV 116 CAT-001SLIEHLQGL 117 DDX60L-001 SLIQHLEEI 118 LRRC70-001 SLLKNLIYL 119PTPLB-001 SLLNHLPYL 120 HDAC5-001 SLLQHVLLL 121 VPS13B-002 SLLQKQIML 122ZNF318-001 SLSQELVGV 123 CCDC51-001 SVLGALIGV 124 IFIT1-001 VLLHHQIGL125 NYESO1-001 SLLMWITQV 126 ACPL-001 LLLVHLIPV 139 HSPB3-001 IILRHLIEI140 UNC7-001 KILLHLIHI 141 SCYL2-001 KVLPHLIPL 142 RPS2P8-001 SALVHLIPV143 PCNXL3-003 NALVHLIEV 144 AQP6-001 VALGHLIGI 145 PCNX-001 NALVHLIEI146 AQP6-002 WALGHLIGI 147 TRGV10-001 QALEHLIYI 148 NECAP1-001 ISLAHLILV149 FBXW2-001 ETLDHLISL 150 ACCSL-001 ALLSHLICR 151 ACER1-001 KELRHLIEV152 ADAMTS14-001 IALVHLIMV 153 ARHGAP17-001 CWLCHLIKL 154 ARSE-001GKLTHLIPV 155 ATP-009 HLLMHLIGS 156 AUNI-001 TQLDHLIPG 157 C16orf96-001QDLWHLIKL 158 CDC7-002 IALKHLIPT 159 CDC7-003 IALKHLILT 160 CHRNA1-001LQLIHLINV 161 FASTKD5-001 SQLVHLIYV 162 FRYL-002 CLLPHLIQH 163 FTH1-001MVLVHLIHS 164 HERC4-002 SDLFHLIGV 165 HPS5-001 KLLFHLIQS 166 HPS5-002KLLLHLIQS 167 HTR2C-001 SFLVHLIGL 168 IPM-001 YGLKHLISV 169 KIF16-001SELPHLIGI 170 KLHL33-001 YALSHLIHA 171 LAMA3-001 TLLGHLISK 172LOC100128170-001 SQLSHLIAM 173 MAP2K7-001 FFLVHLICM 174 MON2-003VSLHHLINA 175 OR2AK2-001 IMLIHLIRL 176 OR2AK2-002 ITLIHLIRL 177OR2B6-001 SELFHLIPL 178 OR2B6-002 SVLFHLIPL 179 OTUD7A-001 AQLAHLILS 180OVOS2-001 FLLGHLIPR 181 PIGC-002 MLLGHLIFF 182 RAD54L2-003 VLLFHLIEE 183RASEF-001 VFLRHLITL 184 RASGRF1-003 TLLDHLIFK 185 RPS2P20-001 SVLVHLIPA186 SACS-001 AKLEHLIYL 187 SPATA31D5-001 SLLPHLILS 188 TPST2-001SILGHLICS 189 TRGV10-002 QSLEHLIYI 190 UGP-001 YILNHLINP 191 USP51-001YKLLHLIWI 192 ZNF423-002 KLLCHLIEH 193 ZNF584-001 ALLDHLITH 194ZNF99-001 FMLSHLIQH 195

EXAMPLES

Seven PRAME-specific TCRs directed to the herein disclosed PRAME-004peptide (R11P3D3, R16P1C10, R16P1E8, R17P1A9, R17P1D7, R17P1G3 andR17P2B6, see Table 1), each encoding tumor specific TCR-alpha andTCR-beta chains, were isolated and amplified from T-cells of healthydonors. Cells from healthy donors were in vitro stimulated according toa method previously described (Walter et al., 2003 J Immunol., November15; 171(10):4974-8) and target-specific cells were single-cell sortedusing HLA-A*02 multimers and then used for subsequent TCR isolation. TCRsequences were isolated via 5′ RACE by standard methods as described bye.g. Molecular Cloning a laboratory manual fourth edition by Green andSambrook. The alpha and beta variable regions of TCRs R11P3D3, R16P1C10,R16P1E8, R17P1A9, R17P1D7, R17P1G3 and R17P2B6 were sequenced and clonedfor further functional characterization.

R11P3D3, R16P1C10, R17P1D7 and R17P2B6 are derived from HLA-A*02negative donor (alloreactive setting) and R16P1E8, R17P1A9 and R17P1G3are derived from a HLA-A*02 positive donor.

Furthermore, the mutant TCR R11P3D3_KE, an enhanced variant of R11P3D3,is herein disclosed. Enhanced TCR variant R11P3D3_KE was modified fromthe parental TCR as described in PCT/EP2017/081745, herewithspecifically incorporated by reference, and in example 8 below, and thecoding sequence was obtained by gene synthesis prior to the functionalcharacterization of the TCR.

Example 1: T-Cell Receptor R11P3D3

TCR R11P3D3 (SEQ ID NO:1-12 and 196) is restricted towardsHLA-A*02-presented PRAME-004 (SEQ ID NO:97) (see FIG. 8).

R11P3D3 specifically recognizes PRAME-004, as human primary CD8+ T-cellsre-expressing this TCR release IFNγ upon co-incubation with HLA-A*02+target cells, loaded either with PRAME-004 peptide or alanine orthreonine substitution variants of PRAME-004 (FIG. 1) or differentpeptides showing high degree of sequence similarity to PRAME-004 (FIG.8). NYESO1-001 peptide is used as negative control. TCR R11P3D3 has anEC₅₀ of 0.74 nM (FIG. 15) and a binding affinity (K_(D)) of 18-26 μMtowards HLA-A*02-presented PRAME-004 (SEQ ID NO:97).

Re-expression of R11P3D3 in human primary CD8+ T-cells leads toselective recognition and killing of HLA-A*02/PRAME-004-presenting tumorcell lines (FIGS. 24, 25 30 and 32). TCR R11P3D3 does not respond to anyof the 25 tested healthy, primary or iPSC-derived cell types (FIGS. 24and 25) and was tested for cross-reactivity towards further 67 similarpeptides (of which 57 were identical to PRAME-004 in positions 3, 5, 6and 7) but unrelated peptides in the context of HLA-A*02 (FIGS. 8, 22and 23).

Example 2: T-Cell Receptor R16P1C10

TCR R16P1C10 (SEQ ID NO:13-24 and 197) is restricted towardsHLA-A*02-presented PRAME-004 (SEQ ID NO:97) (see FIG. 9).

R16P1C10 specifically recognizes PRAME-004, as human primary CD8+T-cells re-expressing this TCR release IFNγ upon co-incubation withHLA-A*02+ target cells and bind HLA-A*02 tetramers (FIG. 21),respectively, loaded either with PRAME-004 peptide or alanine orthreonine substitution variants of PRAME-004 (FIG. 2) or differentpeptides showing high degree of sequence similarity to PRAME-004 (FIG.9). NYESO1-001 peptide is used as negative control. TCR R16P1C10 has anEC₅₀ of 9.6 nM (FIG. 16).

Example 3: T-Cell Receptor R16P1E8

TCR R16P1E8 (SEQ ID NO:25-36 and 198) is restricted towardsHLA-A*02-presented PRAME-004 (SEQ ID NO:97) (see FIG. 10).

R16P1E8 specifically recognizes PRAME-004, as human primary CD8+ T-cellsre-expressing this TCR release IFNγ upon co-incubation with HLA-A*02+target cells, loaded either with PRAME-004 peptide or alanine orthreonine substitution variants of PRAME-004 (FIG. 3) or differentpeptides showing high degree of sequence similarity to PRAME-004 (FIG.10). NYESO1-001 peptide is used as negative control. TCR R16P1E8 has anEC₅₀ of ˜1 μM (FIG. 17).

Example 4: T-Cell Receptor R17P1A9

TCR R17P1A9 (SEQ ID NO:37-48 and 199) is restricted towardsHLA-A*02-presented PRAME-004 (SEQ ID NO:97) (see FIG. 11).

R17P1A9 specifically recognizes PRAME-004, as human primary CD8+ T-cellsre-expressing this TCR release IFNγ upon co-incubation with HLA-A*02+target cells, loaded either with PRAME-004 peptide or alaninesubstitution variants of PRAME-004 (FIG. 4) or different peptidesshowing high degree of sequence similarity to PRAME-004 (FIG. 11).NYESO1-001 peptide is used as negative control.

Example 5: T-Cell Receptor R17P1D7

TCR R17P1D7 (SEQ ID NO:49-60 and 200) is restricted towardsHLA-A*02-presented PRAME-004 (SEQ ID NO:97) (see FIG. 12).

R17P1D7 specifically recognizes PRAME-004, as human primary CD8+ T-cellsre-expressing this TCR release IFNγ upon co-incubation with HLA-A*02+target cells, loaded either with PRAME-004 peptide or alanine orthreonine substitution variants of PRAME-004 (FIG. 5) or differentpeptides showing high degree of sequence similarity to PRAME-004 (FIG.12). NYESO1-001 peptide is used as negative control. TCR R17P1D7 has anEC₅₀ of 1.83 nM (FIG. 18).

Example 6: T-Cell Receptor R17P1G3

TCR R17P1G3 (SEQ ID NO:61-72 and 201) is restricted towardsHLA-A*02-presented PRAME-004 (SEQ ID NO:97) (see FIG. 13).

R17P1G3 specifically recognizes PRAME-004, as human primary CD8+ T-cellsre-expressing this TCR release IFNγ upon co-incubation with HLA-A*02+target cells, loaded either with PRAME-004 peptide or alanine orthreonine substitution variants of PRAME-004 (FIG. 6) or differentpeptides showing high degree of sequence similarity to PRAME-004 (FIG.13). NYESO1-001 peptide is used as negative control. TCR R17P1G3 has anEC₅₀ of 8.63 nM (FIG. 19).

Example 7: T-Cell Receptor R17P2B6

TCR R17P2B6 (SEQ ID NO:73-84 and 202) is restricted towardsHLA-A*02-presented PRAME-004 (SEQ ID NO:97) (see FIG. 14).

R17P2B6 specifically recognizes PRAME-004, as human primary CD8+ T-cellsre-expressing this TCR release IFNγ upon co-incubation with HLA-A*02+target cells, loaded either with PRAME-004 peptide or alanine orthreonine substitution variants of PRAME-004 (FIG. 7) or differentpeptides showing high degree of sequence similarity to PRAME-004 (FIG.14). NYESO1-001 peptide is used as negative control. TCR R17P2B6 has anEC₅₀ of 2.11 nM (FIG. 20) and a binding affinity (K_(D)) of 13 μMtowards HLA-A*02-presented PRAME-004.

Example 8: Enhanced T-Cell Receptor R11P3D3_KE

The mutated “enhanced pairing” TCR R11P3D3_KE is introduced as a variantof R11P3D3, where α and β variable domains, naturally bearing αW44/βQ44,have been mutated to αK44/βE44. The double mutation is selected amongthe list present in PCT/EP2017/081745, herewith specificallyincorporated by reference. It is specifically designed to restore anoptimal interaction and shape complementarity to the TCR scaffold.

Compared with the parental TCR R11P3D3 the enhanced TCR R11P3D3_KE showssuperior sensitivity of PRAME-004 recognition. The response towardsPRAME-004-presenting tumor cell lines are stronger with the enhanced TCRR11P3D3_KE compared to the parental TCR R11P3D3 (FIG. 30). Furthermore,the cytolytic activity of R11P3D3_KE is stronger compared to R11P3D3(FIG. 32). The observed improved functional response of the enhanced TCRR11P3D3_KE is well in line with an increased binding affinity towardsPRAME-004, as described in example 1 (R11P3D3, K_(D)=18-26 μM) andexample 8 (R11P3D3_KE, K_(D)=5.3 μM).

The invention claimed is:
 1. A method of treating a patient who has aPRAME positive cancer, comprising administering to the patient apopulation of transformed CD8+ T cells expressing at least one vectorencoding a T cell receptor (TCR), wherein the TCR comprises a CDR1αchain comprising the amino acid sequence of SEQ ID NO: 1, a CDR2α chaincomprising the amino acid sequence of SEQ ID NO: 2, a CDR3α chaincomprising the amino acid sequence of SEQ ID NO: 3, a CDR1β chaincomprising the amino acid sequences of SEQ ID NO: 7, a CDR2β chaincomprising the amino acid sequence of SEQ ID NO: 8, and a CDR3β chaincomprising the amino acid sequence of SEQ ID NO: 9, wherein the TCR iscapable of binding to a peptide consisting of the amino acid sequence ofSLLQHLIGL (SEQ ID NO: 97) in a complex with HLA-A*02, and wherein thecancer is selected from acute lymphocytic cancer, acute myeloidleukemia, alveolar rhabdomyosarcoma, bone cancer, brain cancer, breastcancer, cancer of the anus, anal canal, or anorectum, cancer of the eye,cancer of the intrahepatic bile duct, cancer of the joints, cancer ofthe neck, gallbladder, or pleura, cancer of the nose, nasal cavity, ormiddle ear, cancer of the oral cavity, cancer of the vagina, cancer ofthe vulva, chronic lymphocytic leukemia, chronic myeloid cancer, coloncancer, esophageal cancer, cervical cancer, gastrointestinal carcinoidtumor, glioma, Hodgkin lymphoma, hypopharynx cancer, kidney cancer,larynx cancer, liver cancer, lung cancer, malignant mesothelioma,melanoma, multiple myeloma, nasopharynx cancer, non-Hodgkin lymphoma,cancer of the oropharynx, ovarian cancer, cancer of the penis,pancreatic cancer, peritoneum, omentum, and mesentery cancer, pharynxcancer, prostate cancer, rectal cancer, renal cancer, skin cancer, smallintestine cancer, soft tissue cancer, stomach cancer, testicular cancer,thyroid cancer, cancer of the uterus, ureter cancer, and urinary bladdercancer.
 2. The method of claim 1, wherein the population of transformedcells are produced by a method comprising isolating a cell from asubject, transforming the cell with at least one vector encoding the TCRto produce a transformed cell, and expanding the transformed cell toproduce the population of transformed cells.
 3. The method of claim 2,wherein the subject is the patient.
 4. The method of claim 2, whereinthe subject is a healthy donor.
 5. A method of treating a patient whohas a PRAME positive cancer, comprising administering to the patient apopulation of transformed CD8+ T cells expressing at least one vectorencoding a T cell receptor (TCR), wherein the TCR comprises an α chaincomprising the amino acid sequence of SEQ ID NO: 6 and a β chaincomprising the amino acid sequence of SEQ ID NO: 12, wherein the TCR iscapable of binding to a peptide consisting of the amino acid sequence ofSLLQHLIGL (SEQ ID NO: 97) in a complex with HLA-A*02, and wherein thecancer is selected from acute lymphocytic cancer, acute myeloidleukemia, alveolar rhabdomyosarcoma, bone cancer, brain cancer, breastcancer, cancer of the anus, anal canal, or anorectum, cancer of the eye,cancer of the intrahepatic bile duct, cancer of the joints, cancer ofthe neck, gallbladder, or pleura, cancer of the nose, nasal cavity, ormiddle ear, cancer of the oral cavity, cancer of the vagina, cancer ofthe vulva, chronic lymphocytic leukemia, chronic myeloid cancer, coloncancer, esophageal cancer, cervical cancer, gastrointestinal carcinoidtumor, glioma, Hodgkin lymphoma, hypopharynx cancer, kidney cancer,larynx cancer, liver cancer, lung cancer, malignant mesothelioma,melanoma, multiple myeloma, nasopharynx cancer, non-Hodgkin lymphoma,cancer of the oropharynx, ovarian cancer, cancer of the penis,pancreatic cancer, peritoneum, omentum, and mesentery cancer, pharynxcancer, prostate cancer, rectal cancer, renal cancer, skin cancer, smallintestine cancer, soft tissue cancer, stomach cancer, testicular cancer,thyroid cancer, cancer of the uterus, ureter cancer, and urinary bladdercancer.
 6. The method of claim 1, wherein the population of transformedcells are administered in the form of a pharmaceutical composition. 7.The method of claim 6, wherein the pharmaceutical composition comprisesa chemotherapeutic agent selected from the group consisting ofasparaginase, busulfan, carboplatin, cisplatin, daunorubicin,doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate,paclitaxel, rituximab, vinblastine, and vincristine.
 8. The method ofclaim 1, wherein the TCR comprises: a CDR1α chain comprising the aminoacid sequence of SEQ ID NO: 1, a CDR2α chain comprising the amino acidsequence of SEQ ID NO: 2, a CDR3α chain consisting of the amino acidsequence of SEQ ID NO: 3, a CDR1β chain comprising the amino acidsequences of SEQ ID NO: 7, a CDR2β chain comprising the amino acidsequence of SEQ ID NO: 8, and a CDR3β chain consisting of the amino acidsequence of SEQ ID NO:
 9. 9. The method of claim 1, wherein the TCRcomprises: a CDR1α chain consisting of the amino acid sequence of SEQ IDNO: 1, a CDR2α chain comprising the amino acid sequence of SEQ ID NO: 2,a CDR3α chain comprising the amino acid sequence of SEQ ID NO: 3, aCDR1β chain consisting of the amino acid sequences of SEQ ID NO: 7, aCDR2β chain comprising the amino acid sequence of SEQ ID NO: 8, and aCDR3β chain comprising the amino acid sequence of SEQ ID NO:
 9. 10. Themethod of claim 1, wherein the TCR comprises a CDR1α chain consisting ofthe amino acid sequence of SEQ ID NO: 1, a CDR2α chain consisting of theamino acid sequence of SEQ ID NO: 2, a CDR3α chain consisting of theamino acid sequence of SEQ ID NO: 3, a CDR1β chain consisting of theamino acid sequence of SEQ ID NO: 7, a CDR2β chain consisting of theamino acid sequence of SEQ ID NO: 8, and a CDR3β chain consisting of theamino acid sequence of SEQ ID NO:
 9. 11. The method of claim 1, whereinthe cancer is melanoma.
 12. The method of claim 1, wherein the cancer isbone cancer.
 13. The method of claim 10, wherein the cancer is melanoma.14. The method of claim 10, wherein the cancer is bone cancer.